The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin(Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63)The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases(Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)Eraldo Antonini (1931–1983) received his degree in Medicine and Surgery from the University of Rome in 1955. He then began studying hemoglobin and myoglobin with Alessandro Rossi-Fanelli at the Institute of Biochemistry of the University of Rome and at the Regina Elena Institute for Cancer Research.Open in a separate windowEraldo Antonini (right) in Caprarola in 1971 for Jeffries Wyman''s (left) birthday party, with Robert W. Noble (back).In 1961, Antonini and Rossi-Fanelli published a paper describing the effect on human hemoglobin''s activity when the C-terminal amino acid residues are removed from the molecule''s α and/or β chains. This paper is reprinted here as a Journal of Biological Chemistry (JBC) Classic. In the study, the scientists used carboxypeptidase A to digest the C-terminal tyrosine and histidine on the molecule''s β chain and carboxypeptidase B to remove the C-terminal lysine, tyrosine, and arginine on the molecule''s α chain. The resulting protein appeared intact but had an increased oxygen affinity, lowered cooperativity, and dramatically reduced Bohr effect.This observation inspired Max Perutz, who wrote: “Several years later, my electron density maps showed that these residues form salt bridges with neighboring subunits in deoxyhaemoglobin which get broken on transition to oxyhaemogloblin. Remembering Antonini''s observation, I realized at once that these bridges must represent the additional bonds between the subunits in the T structure predicted by Monod, Wyman and Changeux''s theory of allostery. Antonini had also demonstrated that the release of Bohr protons is colinear with oxygen uptake. When Kilmartin''s and my work proved that most of the Bohr protons originate from the salt bridges, it became clear to me that oxygen uptake is linked to the rupture of these bridges” (1).In the second JBC Classic reprinted here, Antonini follows up on the first paper by doing a reciprocal experiment in which he looks at differences in digestion rates of oxy- and deoxyhemoglobin, reasoning, “If enzymatic modification can affect conformation and changes of conformation resulting from combination with ligand (oxygen), one might expect that the rate of attack on the hemoglobin by the enzymes should depend on the presence or absence of ligand; this would determine conformation, and conformation, in turn, would control the rate.” Again using carboxypeptidases A and B, he showed that the rate of digestion is different for the oxy- and deoxy- forms of the molecule, indicating a differential accessibility of the C-terminal residues to these enzymes.This work was later extended and perfected by John V. Kilmartin on a suggestion by Perutz, who pointed out the crucial role of the C-terminal residues for the molecular mechanism of cooperativity and the Bohr effect. Kilmartin was able to differentiate the role of the C-terminal histidine from that of tyrosine by preparing and characterizing a modified hemoglobin devoid of histidine.Over the next several years, Antonini continued to study hemoglobin, looking at the properties of the α and β chains, the acid-base equilibria of hemoglobin, the Bohr effect and its dependence on temperature, the oxidation-reduction equilibria, ligand-induced conformational changes in hemoglobin, and the kinetics of the reaction of myoblogins and hemoglobins with ligands. This work culminated in the publication of Hemoglobin and Myoglobin in Their Reactions with Ligands in 1971 (2), which was a landmark in the field.In the 1970s, Antonini expanded his scientific interests and started focusing on electron-transfer metalloproteins (such as cytochrome c oxidase) and on proteolytic enzymes. He eventually became Professor of Molecular Biology at the University of Camerino and was later made Professor of Chemistry and Director of the Institute of Chemistry in the Faculty of Medicine and Surgery at the University of Rome. He also received the Feltrinelli Prize from the Accademia Nazionale dei Lincei in 1974.One of Antonini''s coauthors on the two JBC Classics reprinted here is JBC Classic author Jeffries Wyman (3) who came to Rome in 1961 for a week-long visit and ended up remaining for 25 years and working with Antonini. This collaboration produced a series of outstanding papers and conceptual advancements that have had a long lasting influence on protein chemistry.^1,2     

The process of reversible phosphorylation: the work of Edmond H. Fischer

Edmond H. Fischer was awarded the 1992 Nobel Prize in Physiology or Medicine for his joint research with Edwin G. Krebs on reversible protein phosphorylation. The two Classics reprinted here relate some of Fischer and Krebs'' early discoveries in their phosphorylase researchPhosphorylase Activity of Skeletal Muscle Extracts (Krebs, E. G., and Fischer, E. H. (1955) J. Biol. Chem. 216, 113–120)Conversion of Phosphorylase b to Phosphorylase a in Muscle Extracts (Fischer, E. H., and Krebs, E. G. (1955) J. Biol. Chem. 216, 121–132)Edmond H. Fischer was born in Shanghai, China in 1920. He was sent to boarding school in Switzerland at age 7, and in 1935, he entered Geneva''s Collège de Calvin. There, he became friends with his classmate Wilfried Haudenschild, and together, they decided that one of them should go into the sciences and the other into medicine so they could cure the world of all ills. Fischer chose science.Open in a separate windowEdmond H. FischerJust before the start of World War II, Fischer completed high school and entered the School of Chemistry at the University of Geneva. He earned two Licences ès Sciences, one in biology, the other in chemistry, and 2 years later, he was awarded a Diploma of “Ingénieur Chimiste.” For his thesis, he worked with Kurt H. Meyer on the purification of amylase from hog and human pancreas, as well as saliva and several strains of bacteria.In 1950, Fischer went to the United States to do a postdoctoral fellowship with Paul Karrer at CalTech. However, when he arrived in Pasadena he received a letter from Journal of Biological Chemistry (JBC) Classic author Hans Neurath (1), chairman of the department of biochemistry at the University of Washington, offering him an assistant professorship in his department. Fischer visited Seattle and accepted the offer, in part because the surrounding mountains, forests, and lakes reminded him of his native Switzerland.Within 6 months of his arrival, Fischer started working on glycogen phosphorylase with Edwin G. Krebs, who was featured in a previous JBC Classic (2). Krebs had trained with JBC Classic authors Carl and Gerty Cori who had discovered that muscle phosphorylase exists in two forms, phosphorylase a, which was easily crystallized and was active without the addition of AMP, and phosphorylase b, a more soluble protein, which was inactive without AMP (3). They believed that AMP served some kind of co-factor function for the enzyme, facilitating its transition between the two forms.However, in Geneva, Fischer had purified potato phosphorylase, which had no AMP requirement. Because it seemed unlikely that muscle phosphorylase but not potato phosphorylase would require AMP as a co-factor, Fischer and Krebs decided to try to elucidate the role of AMP in the phosphorylase reaction. They never discovered what the nucleotide was doing (this problem was solved several years later when Jacques Monod proposed his allosteric model for the regulation of enzymes), but they did discover that muscle phosphorylase was regulated by an enzyme-catalyzed phosphorylation-dephosphorylation reaction.The two JBC Classics reprinted here relate some of Fischer and Krebs'' early discoveries in their phosphorylase research. In the first Classic, the pair performed experiments to determine whether environmental temperature affects the phosphorylase content of skeletal muscle. They were unable to detect any temperature effects, but they did make the surprising discovery that the muscle extracts contained mainly phosphorylase b rather than phosphorylase a. The pair concluded that “If resting muscle contains mainly phosphorylase b… then pronounced activation of the phosphorylase reaction under various conditions is possible.”The second JBC Classic was printed back-to-back with the first. In it, Krebs and Fischer examine the requirements for the phosphorylase conversion and present evidence that the conversion of phosphorylase b to a in cell-free muscle extracts requires a nucleotide containing high energy phosphate and a divalent metal ion. However, they state that “whether this implies that during conversion there is a direct phosphorylation of the enzyme or the formation of an ‘active’ intermediate cannot be stated at this time. It is also possible that the function of ATP is concerned with the synthesis of a prosthetic group.”Similar work was being carried out on liver phosphorylase at approximately the same time by Earl Sutherland. As discussed in a previous JBC Classic (4), Sutherland discovered the second messenger cyclic AMP (cAMP), which he showed promoted the phosphorylation and activation of phosphorylase. The way in which cAMP promoted phosphorylase activation was eventually elucidated when Krebs and Fischer discovered phosphorylase kinase, which was responsible for phosphorylating phosphorylase. Phosphorylase kinase itself existed in a highly activated phosphorylated form and a less active nonphosphorylated form.As a result of the significance of their studies, Krebs and Fischer were awarded the 1992 Nobel Prize in Physiology or Medicine “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism.”In addition to the Nobel Prize, Fischer has received many awards and honors in recognition of his contributions to science. These include the Werner Medal from the Swiss Chemical Society, the Lederle Medical Faculty Award, the Prix Jaubert from the University of Geneva, and jointly with Krebs, the Senior Passano Award and the Steven C. Beering Award from Indiana University. Fischer was elected to the American Academy of Arts and Sciences in 1972 and to the National Academy of Sciences in 1973.¹     

Succeeding in Science Despite the Odds; Studying Metabolism with NMR by Mildred Cohn

A Study of Oxidative Phosphorylation with O¹⁸-labeled Inorganic Phosphate(Cohn, M. (1953) J. Biol. Chem. 201, 735–750)Nuclear Magnetic Resonance Spectra of Adenosine Di- and Triphosphate. II. Effect of Complexing with Divalent Metal Ions(Cohn, M., and Hughes, T. R. (1962) J. Biol. Chem. 237, 176–181)Mildred Cohn was born in New York City in 1913. When she was young, her father told her she could achieve anything she chose to, but not without some difficulty because she was both female and Jewish. With her parents'' encouragement, Cohn moved rapidly through the New York public school system and graduated from high school at age 14. She decided to go to Hunter College in Manhattan, then an all-girls college, and majored in chemistry and minored in physics. Hunter''s attitude toward science education at that time can be summed up by the chairman of the chemistry department who declared that it was not ladylike for women to be chemists and that his sole purpose was to prepare his students to become chemistry teachers.When Cohn graduated from Hunter College in 1931 she tried to get a scholarship for graduate studies in chemistry but was unsuccessful. She enrolled in Columbia University nonetheless and used her savings to pay for her education. At Columbia, she studied under Nobel laureate Harold Urey but had to drop out after a year because of lack of money. She then took a job with the National Advisory Committee of Aeronautics and after a few years was able to earn enough money to return to Columbia. Working with Urey, she studied ways of separating different isotopes of carbon and received her Ph.D. in physical chemistry in 1937.Unfortunately, jobs were scarce in 1938, during the years of the Great Depression, and academic positions for women were even more scarce. Industrial recruiters regularly posted notices announcing that, “Mr. X of Y Company will interview prospective doctorate recipients—Male, Christian”(1).¹ With Urey''s help, Cohn was able to obtain a postdoctoral position at George Washington University with future Nobel prize winner Vincent du Vigneaud. In du Vigneaud''s laboratory, Cohn pioneered the effort to use isotopic tracers to follow the metabolism of sulfur-containing compounds, the subject of a previous Journal of Biological Chemistry (JBC) Classic (2). Cohn worked with du Vigneaud for 9 years and moved with him to New York when he went to Cornell Medical College.In 1946, Cohn went to Washington University in St. Louis to work with Carl and Gerty Cori, Nobel prize laureates and authors of a previous JBC Classic (3), who were studying biological catalysts. There, she did independent research, mainly focusing on using isotopes and NMR to study metabolic processes. Cohn was promoted to Associate Professor in Biochemistry in 1958 but left Washington University 2 years later to move to the University of Pennsylvania School of Medicine. She became a full Professor in 1961 and retired as Benjamin Rush Professor Emerita of Biochemistry and Biophysics in 1982.Once, when asked what her most exciting scientific moments were (4), Cohn replied, “In 1958, using nuclear magnetic resonance, I saw the first three peaks of ATP (5). That was exciting. [I could] distinguish the three phosphorus atoms of ATP with a spectroscopic method, which had never been done before. Another paper, in 1962 (the second JBC Classic reprinted here), was about the effect of metal ions on the phosphorus spectrum of ATP. And earlier, I found that oxygen in inorganic phosphate exchanged with water through oxidative phosphorylation (the first JBC Classic reprinted here).”Cohn''s study of oxidative phosphorylation came at a time when it was known that phosphorylation occurred concomitantly with oxidation in the electron transport chain. However, no one had yet discovered the nature of the interaction of the electron transport system with phosphate or any part of the phosphorylating system. Cohn approached this problem by tracking the loss of O¹⁸ from inorganic phosphate during oxidative phosphorylation in rat liver mitochondria. In the first JBC Classic reprinted here, she describes her findings as, “a new reaction which occurs in oxidative phosphorylation associated with the electron transport system has been observed in the rat liver mitochondria with α-ketoglutarate, â-hydroxybutyrate, and succinate as substrates. This reaction manifests itself by a replacement of O¹⁸ with normal O¹⁶ in inorganic phosphate labeled with O¹⁸ and parallels the phosphorylation which is associated with the oxidation.” Cohn concluded that water must be involved in this reaction because there was no other source of oxygen large enough to account for the amounts she saw introduced into inorganic phosphate. In the second JBC Classic Cohn describes her use of NMR to examine the structural changes in ADP and ATP caused by various divalent metal ions. Cohn knew that divalent ions were involved in enzymatic reactions of ADP and ATP but didn''t know their functions. Using NMR, she measured the changes in the chemical shifts in the peaks of the ATP and ADP phosphorus nuclei in the presence of Mg²⁺, Ca²⁺, and Zn²⁺ as well as the paramagnetic ions Cu²⁺, Mn²⁺, and Co²⁺. By analyzing the resultant spectra, she was able to determine which metals bound to which phosphate groups and thus gained insight into the nature of the metal complexes formed.Cohn received many awards and honors for her contributions to science, including the National Medal of Science in 1982, “for pioneering the use of stable isotopic tracers and nuclear magnetic resonance spectroscopy in the study of the mechanisms of enzymatic catalysis,” as well as election to the National Academies of Science in 1971. She also served the American Society of Biological Chemists (ASBC), now American Society for Biochemistry and Molecular Biology (ASBMB), in many ways. She was President of the Society in 1978 and was on the Federation of American Societies for Experimental Biology (FASEB) Board from 1978 to 1980 as ASBC representative. In addition, Cohn was the first woman to be appointed to the JBC Editorial Board.Despite her success, Cohn''s father was right about the difficulties she would encounter in her life. “My career has been affected at every stage by the fact that I am a woman, beginning with my undergraduate education, which was very inferior in chemistry, and physics was not even offered [as a major] at Hunter College, unlike the excellent science education that my male counterparts received at City College,” she notes. “In my day, I experienced discrimination in academia, government, and industry.”     

DNA Repair Mechanisms: the Work of Aziz Sancar

Action Mechanism of Escherichia coli DNA Photolyase. III. Photolysis of the Enzyme-Substrate Complex and the Absolute Action Spectrum(Sancar, G. B., Jorns, M. S., Payne, G., Fluke, D. J., Rupert, C. S., and Sancar, A. (1987) J. Biol. Chem. 262, 492–498)Reconstitution of the Human DNA Repair Excision Nuclease in the Highly Defined System(Mu, D., Park, C.-H., Matsunaga, T., Hsu, D. S., Reardon, J. T., and Sancar, A. (1995) J. Biol. Chem. 270, 2415–2418)Aziz Sancar was born in Savur, Turkey in 1946. Although both his parents were illiterate, they valued the importance of education and did their best to see that Sancar received a good one. They succeeded, and he excelled in many scientific subjects in high school. However, he also dreamed of playing on Turkey''s national soccer team, and this dream almost came true when, as a senior in high school, he was invited to attend tryouts to be a goalie on the national under-18 team. Ultimately he decided not to accept the invitation, later explaining, “upon serious consideration, I decided I wasn''t tall enough to be an outstanding goalie, and instead I concentrated on my studies” (1).Open in a separate windowAziz SancarAfter graduating in 1963, Sancar enrolled at Istanbul Medical School with the idea of becoming a doctor. However, after taking a biochemistry class during his 2nd year, he decided to become a research biochemist. Surprisingly, when he discussed his desire to pursue a Ph.D. with his biochemistry professor, he advised Sancar to practice medicine briefly before plunging into research, reasoning that anyone who spends the time getting a medical degree should at least practice for a couple years. So, Sancar spent 2 years as a rural physician near his hometown of Savur.In 1973, Sancar came to the United States to study with Claud Rupert in the molecular biology department of the University of Texas at Dallas. While in Turkey, Sancar had developed an interest in photoreactivation, the process by which DNA damaged by UV light is repaired by longer wavelength blue light. Rupert had identified photolyase, the enzyme that mediated the process by catalyzing the opening of the cyclobutane ring in pyrimidine dimers, and Sancar was eager to work him. The main topic of study in the Rupert laboratory in the early 1970s was the nature of photolyase''s chromophore. To that end, Sancar spent several years cloning and characterizing the gene for photolyase (2). After finally succeeding, he set out to purify the protein, but Rupert told him he had done enough research for his thesis and advised him to write his dissertation and graduate.After earning his Ph.D. in 1977, Sancar applied to three different laboratories hoping to continue studying DNA repair. All three laboratories rejected him. However, he learned that Dean Rupp at Yale University was interested in cloning repair genes, and although he didn''t have a postdoctoral position available, he was looking for a technician. Sancar accepted the job and joined the lab. Working with Rupp, Sancar identified and cloned several Escherichia coli repair genes, including the uvrA, uvrB, and uvrC genes involved in excision repair (3–5). He then purified the three uvr proteins and reconstituted the UVRABC nuclease, which he termed “excision nuclease” or “excinuclease” (6).In 1982, Sancar left Yale to become an associate professor of biochemistry at the University of North Carolina, Chapel Hill. There he resumed his work on photolyase and discovered that the enzyme contains two chromophores: FADH⁻ and a pterin (7–9). He also proposed a model for the reaction mechanism of photolyase repair, which is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here.At the time the Classic was published, there were two possible mechanisms for the repair reaction: the first involved energy transfer from a sensitizer to pyrimidine dimers, and the second involved electron transfer between the pyrimidine dimer and the photosensitizer. By determining the absolute action spectrum of the enzyme, Sancar and his colleagues were able to determine that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300–500 nm range, the photolyase chromophore donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. Eighteen years after publishing this Classic paper, Sancar was able to capture the excited flavin intermediate and observe the photolyase electron transfer, definitively proving his model (10).Sancar also continued studying other DNA repair pathways and soon turned his attention to excision repair in humans. The second JBC Classic is a result of Sancar''s studies on xeroderma pigmentosum, a hereditary disease caused by a defect in nucleotide excision repair as a result of mutations in one of several genes: XPA through XPG. In the paper, Sancar and his colleagues purified the components known to be required for the incision reaction and reconstituted the excision nuclease activity with these proteins. Using this system, they determined that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.Sancar is currently the Sarah Graham Kenan Professor of Biochemistry and Biophysics at the UNC School of Medicine. He has received many honors and awards in recognition of his contributions to science, including the Presidential Young Investigator Award from the National Science Foundation (1984) and the highest awards from the American Society for Photobiology (1990) and the Turkish Scientific Research Council (1995). Sancar was also the first Turkish-American member of the National Academy of Sciences (2005).     

The Role of Binding Energy in Catalysis: the Work of William P. Jencks

Two Functional Domains of Coenzyme A Activate Catalysis by Coenzyme A Transferase. Pantetheine and Adenosine 3′-Phosphate 5′-Diphosphate (Fierke, C. A., and Jencks, W. P. (1986) J. Biol. Chem. 261, 7603–7606)William Platt Jencks (1927–2007) was born in Bar Harbor, Maine. He became interested in chemistry when he received a chemistry set for Christmas in 1934. He immediately carried out one of the experiments described in the instructions, the addition of dilute acid to a sulfide salt to produce H₂S. The experiment was so successful that his house had to be evacuated due to the smell of rotten eggs. According to Jencks, “My family and I did not find it necessary to replicate this experiment” (1).Open in a separate windowWilliam P. JencksJencks enrolled at Harvard College, intending to study chemistry. However, after taking a first year course in chemistry that “described a large number of chemical reactions, one after the other, with no indication of what was interesting about any of them” (1), he switched his major to English. Despite this change in the direction of his studies, Jencks ended up entering Harvard Medical School after his junior year because he wasn''t sure what else to do.After completing his first year of medical school, Jencks spent a summer at the Marine Biological Laboratory in Woods Hole, taking courses and doing research on lobster shell pigments with Journal of Biological Chemistry (JBC) Classic author George Wald (2). He received his M.D. in 1951 and then interned at Peter Bent Brigham Hospital in Boston. However, after a while, Jencks found medicine to be “a very broad field in which it would be difficult to obtain definitive answers to fundamental problems” (1). Wald suggested Jencks try doing research at Massachusetts General Hospital with Nobel laureate Fritz Lipmann (who was featured in a previous JBC Classic (3)). Jencks ended up spending 2 years with Lipmann, studying coenzyme A transferase, which led to his longtime interest in the physical organic chemistry of acyl transfer reactions. After leaving Massachusetts General Hospital, Jencks spent a year doing postdoctoral studies at Harvard University with Nobel laureate Robert Woodward before joining the faculty at Brandeis University in 1957, serving as assistant, associate, and then full professor of biochemistry. He retired in 1996 as professor emeritus of biochemistry.During his 39 years at Brandeis University, Jencks studied the mechanisms by which enzymes facilitate chemical reactions of molecules that are not otherwise inclined to react at a useful rate.The JBC Classic reprinted here looks at the noncovalent interactions between succinyl-CoA 3-ketoacid coenzyme A transferase and coenzyme A. In the paper, Jencks and Carol A. Fierke used a small coenzyme A analog, methylmercaptopropionate, to show that noncovalent interactions between the enzyme and the side chain of CoA are responsible for the reaction rate increase brought about by the enzyme. They report that interaction between the enzyme and the pantetheine moiety of CoA provides the majority of substrate destabilization and rate acceleration, whereas the interaction with the 3′-phospho-ADP1 moiety provides binding energy that overcomes this destabilization and permits significant binding of acyl-CoA substrates to the enzyme. This paper helped to illuminate a striking example of the role of binding energy in catalysis.Jencks received many honors and awards for his contributions to science, including memberships in the National Academy of Sciences (1971) and the American Philosophical Society (1995) and foreign membership in the Royal Society. He also received the 1962 American Chemical Society (ACS) Award in Biological Chemistry, the 1993 American Society of Biological Chemists Award, the 1995 ACS James Flack Norris Award in Physical Organic Chemistry, and the 1996 ACS Repligen Award for Chemistry of Biological Processes.¹     

Exploring the world of phospholipids and their interactions with proteins: the work of William Dowhan

The Gene Encoding the Phosphatidylinositol Transfer Protein Is Essential for Cell Growth (Aitken, J. F., van Heusden, G. P., Temkin, M., and Dowhan, W. (1990) J. Biol. Chem. 265, 4711–4717)A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein (Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 271, 11615–11618)William Dowhan''s curiosity about the connections between phospholipids and proteins associated with them goes back as far as his days as a graduate student with Esmond Snell at the University of California, Berkeley. In these two JBC Classics, his group''s ability to manipulate biochemical and molecular genetics tools to answer fundamental questions about lipid biology shines through. “William Dowhan and his research group have made many contributions to the biochemistry of phospholipid metabolism and membrane biogenesis,” says Robert Simoni at Stanford University.Open in a separate windowBill Dowhan (right) is shown here with the late Chris Raetz (left), who was a longtime collaborator and friend, and his former postdoctoral advisor, the late Gene Kennedy, on the occasion of Kennedy''s 90th birthday in 2009 (photo courtesy of William Dowhan).The first paper, published in 1990, documented the importance of phosphatidylinositol/phosphatidylcholine transfer proteins in vivo. Dowhan''s group, which has been based at the University of Texas Medical School since 1972, used a combination of biochemistry and genetics to clone the protein''s gene. Dowhan had first heard of phospholipid transfer proteins in 1969, when he began his postdoctoral training with Eugene (Gene) Kennedy at Harvard Medical School. At his very first Kennedy lab meeting, the discussion centered around a publication that had just come out (1). The paper described “one of the first observations of proteins in the soluble phase that transferred lipids between bilayers,” recalls Dowhan. “No one could figure out what these proteins really did in vivo, but they knew the proteins had this function” of transferring lipids between membranes.As he moved through his career, Dowhan focused on cloning and characterizing genes and purifying enzymes responsible for phospholipid metabolism in Escherichia coli. Then came a sabbatical in 1983 with Gottfried (Jeff) Schatz at the Biozentrum of the University of Basel in Switzerland, that expanded Dowhan''s research directions into yeast genetics. He says the opportunity to work with Schatz “got me into the possibility of looking for this phosphatidylinositol/phosphatidylcholine transfer protein (PI-TP) in yeast, which I probably would have never done if I hadn''t taken this sabbatical.”Fresh from his sabbatical, Dowhan started tracking down the protein and its gene in vivo. “I submitted a grant at that time with some preliminary data that we had begun to purify to homogeneity the PI-TP from yeast, which had never been done before. Fortunately, we got the grant,” he says.The Dowhan group managed to purify PI-TP from yeast. “The most important part was using basic biochemistry and understanding how to purify proteins before the advent of genetically tagging proteins for affinity chromatography,” explains Dowhan.For the next step in the process of finding the gene for the protein, Dowhan and colleagues had to apply reverse genetics because the yeast genome was not available in the late 1980s. They sequenced the amino terminus of the protein, made the corresponding oligonucleotide probes, tested yeast cDNA libraries with those probes, and pulled out the gene. “We still didn''t know the role PI-TP played in cell function. But now we had the sequence of the gene and the knock-out mutant was not viable,” notes Dowhan. “So we published” the findings.At the same time, Vytas Bankaitis, now at the University of North Carolina, had been working on cloning the SEC14 gene in yeast, which is necessary for vesicular transport. “It turns out we had missed the DNA sequence,” Dowhan says. From Bankaitis'' work, it was obvious that “PI-TP was the product of the SEC14 gene. It all came together in a joint report in Nature. Now we had a function associated with the SEC14 gene, which we didn''t have before,” Dowhan explains (2). “We had a phenotype of a mutant lacking this phospholipid transfer protein, which then stopped vesicular transport.”This initial link between phospholipid metabolism and vesicular transport opened up the field to characterization of the Sec14 protein superfamily in a broad range of biological systems. These proteins contain lipid-binding domains, which sense membrane lipid composition and integrate lipid metabolism and lipid-mediated signaling with an array of cellular processes.The second JBC Classic focused on a different feature of phospholipids: their role in protein folding. Dowhan was fascinated by membrane proteins ever since he was a graduate student and had gone to the Kennedy laboratory as a postdoctoral fellow, intending to purify the membrane component expressed by the lac operon for lactose transport in E. coli. He was unsuccessful because, at that time, the necessary detergents were not available. Once the lactose permease was purified (3), Dowhan noticed in the literature that other researchers mentioned that when the protein was reconstituted in liposomes missing phosphatidylethanolamine, the protein was defective in energy-dependent uphill transport. Dowhan recalls that he wondered, “Was that an artifact of the liposome system or was that also true in vivo?”To get to the bottom of this observation, Dowhan''s group used E. coli to generate null mutants of what were considered to be absolutely essential genes for phospholipid synthesis and cell viability. They created a null mutant of the pssA gene, which encodes the committed step to the synthesis of the major phospholipid, phosphatidylethanolamine. By establishing conditions in which bacterial cells lacking phosphatidylethanolamine remained viable, the investigators were able to identify and characterize different cell phenotypes caused by the missing phospholipid both in vivo and in vitro. In collaboration with Ronald Kaback at UCLA, Dowhan''s group showed that phosphatidylethanolamine was essential for the proper folding of an epitope of lactose permease that was also necessary to support the energy-dependent uphill transport of lactose. “Studies by others have since shown a similar chaperone role for lipids in other bacteria, plants and mammalian cells,” notes Simoni.To obtain their data, the investigators developed a new technique, the Eastern-Western blot. In this method, membrane proteins were delipidated and partially denatured by SDS. The proteins underwent gel electrophoresis and then were transferred to a solid support by Western blotting. A series of individual lipids were then laid over the proteins at a 90° angle so that the investigators could see, after incubating with conformation-specific antibodies, which lipids helped which membrane proteins regain proper conformation.This technique was used to establish that phosphatidylethanolamine was necessary in a late step of folding of lactose permease, but was not necessary to maintain the final folded state. This observation suggested that lipids act as molecular chaperones in helping protein maturation. “This paper set the stage for understanding how lipids affect the topological organization of wild-type proteins in the membrane,” notes Dowhan.Dowhan and his collaborator Mikhail Bogdanov have continued using bacterial mutants in phospholipid metabolism to systematically manipulate the native membrane lipid compositions during the cell cycle. They have analyzed the transmembrane domain orientation of membrane proteins to establish the molecular basis for lipid-dependent organization of lactose permease and other secondary transporters (4).Dowhan says his work has two take-home messages. One is that “Lipids aren''t just glorified biological detergents,” he says. “They have specific roles” in the cell. The other message is in the power of numbers. Dowhan says the more techniques applied to solve a biological mystery, the more likely the mystery will be successfully solved.     

The Discovery and Characterization of Molybdopterin: the Work of K. V. Rajagopalan

Hepatic Sulfite Oxidase. A Functional Role for Molybdenum(Cohen, H. J., Fridovich, I., and Rajagopalan, K. V. (1971) J. Biol. Chem. 246, 374–382)Characterization of the Molybdenum Cofactor of Sulfite Oxidase, Xanthine, Oxidase, and Nitrate Reductase. Identification of a Pteridine as a Structural Component(Johnson, J. L., Hainline, B. E., and Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 1783–1786)The Structure of the Molybdenum Cofactor. Characterization of Di(carboxamidomethyl)molybdopterin from Sulfite Oxidase and Xanthine Oxidase(Kramer, S. P., Johnson, J. L., Ribeiro, A. A., Millington, D. S., and Rajagopalan, K. V. (1987) J. Biol. Chem. 262, 16357–16363)K. V. Rajagopalan was born in 1930 in Udupi, a town in South India. He attended Presidency College in Madras (now called Chennai) and graduated with a B.Sc. in Chemistry in 1951. He then enrolled at the University of Madras to do graduate work in the biochemistry department and earned his Ph.D. in 1957. After graduating, Rajagopalan served as an assistant research officer at the Indian Council of Medical Research. Two years later, he started a postdoctoral fellowship in the biochemistry department at Duke University, working with Journal of Biological Chemistry (JBC) Classic author Philip Handler (1).Open in a separate windowK. V. RajagopalanRajagopalan''s initial research project at Duke, carried out in collaboration with JBC Classic author Irwin Fridovich (2), dealt with the competitive inhibition of several enzymes by urea and guanidine. Next, Rajagopalan moved on to studies of aldehyde oxidase and xanthine oxidase and noticed that both enzymes had unusual absorption spectra. Further investigation revealed that they both contained a molybdenum cofactor, something that had not been seen in any other mammalian proteins. Some of Rajagopalan''s electron paramagnetic resonance (EPR) studies on aldehyde oxidase can be found in the JBC Classic on Helmut Beinert (3).These early findings led to an interest in proteins containing molybdenum and formed the basis of Rajagopalan''s future research. In the first JBC Classic reprinted here, Rajagopalan, Fridovich, and Harvey J. Cohen report the discovery of another molybdenum-containing enzyme, sulfite oxidase. The paper documents the presence and function of molybdenum in sulfite oxidase and describes some aspects of its EPR signal. The paper was published along with two other papers (4, 5), which discussed the purification and properties of sulfite oxidase from bovine liver and the nature of its heme prosthetic group.By 1980, several more molecules had been added to the list of molybdenum-containing proteins, but the nature of the cofactor still remained elusive. This was, in part, due to the fact that the activated cofactor was extremely labile in the presence of oxygen and thus very hard to characterize. To circumvent this problem, Rajagopalan developed a method to isolate the oxidized, inactive form of the molybdenum cofactor from sulfite oxidase, xanthine oxidase, and nitrate reductase. His procedure and initial characterization are reported in the second JBC Classic reprinted here. In the paper, Rajagopalan and his colleagues also provided evidence that a pteridine moiety acted as a structural component of the cofactor.Continuing with this work, Rajagopalan was able to isolate two additional stable degradation products (6) and confirm that the molybdenum cofactor consisted of a complex between molybdenum and a unique pterin which he named molybdopterin.In the final JBC Classic reprinted here, Rajagopalan describes the successful isolation of a stable alkylated derivative of molybdopterin, camMPT, from sulfite oxidase and xanthine oxidase by a procedure involving treatment with iodoacetamide under mild denaturing conditions. Structural studies on the product confirmed that molybdopterin is a 6-alkylpterin with a 4-carbon side chain, which has an enedithiol at carbons 1′ and 2′, a hydroxyl at carbon 3′, and a terminal phosphate group.Today, Rajagopalan remains at Duke as James B. Duke Professor of Biochemistry, a position he assumed in 1995. It is interesting to note that, in 1969 when Handler left Duke to become the President of the National Academy of Sciences, he designated Rajagopalan as PI of his National Institutes of Health (NIH) grant. This has subsequently become the longest continuously funded NIH grant, currently in its 62nd year.     

Mycobacterial Glycophosphoinositides: the Work of Clinton E. Ballou

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family of Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046–3056)Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acylation of the Dimannophosphoinositides(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 2975–2984)Clinton Edward Edgerton Ballou was born in King Hill, Idaho in 1923. After graduating from high school, he enrolled in a premed program at Boise Junior College, but his interests quickly turned to chemistry after he dissected a poorly embalmed cat in a comparative anatomy course. During his sophomore year, Ballou transferred to Oregon State College in Corvallis where he became involved in two research projects: the first during his sophomore year synthesizing new antimalarial drugs with Bert Christensen, and the second during his senior year studying the guinea pig “antistiffness factor” with Willem van Wagtendonk.Open in a separate windowClinton E. BallouThe military draft was in effect as Ballou entered his last year of college so he decided to join the U. S. Navy after graduating in 1944. He was discharged 2 years later and decided to apply for graduate study in biochemistry with Karl Paul Link at the University of Wisconsin-Madison. As detailed in a previous Journal of Biological Chemistry (JBC) Classic (1), Link''s research centered on blood anticoagulants, and when Ballou arrived in his laboratory in 1946, the primary focus was the structure-function relationship of coumarin anticoagulants. Ballou was immediately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarol because the acetylated intermediate was degraded in the alkali conditions used for deacetylation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Ballou decided to study a variety of synthetic compounds to try to understand the structural basis for alkali sensitivity. This research formed the core of his doctoral dissertation, and his exposure to carbohydrate chemistry influenced the direction of his career.After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L. Hirst in the Department of Chemistry at the University of Edinburgh. There he studied the structure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to work with Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at the University of California, Berkeley. Ballou explained his choice: “I was attracted to Fischer in part because of his research on phosphorylated sugars but also because during graduate school I had drawn heavily on the published works of his father, Emil Fischer. I guess the idea of being associated with the son of Emil Fischer just seemed ‘real cool’ to me” (2).The 1950s was a time of active research on biosynthetic pathways involving short chain phosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou undertook the syntheses of several such metabolic intermediates, including d-glyceric acid 2-phosphate, d-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate, and d-erythrose 4-phosphate. He also became interested in inositol chemistry as a result of studies on the cyclitols in sugar pine heartwood.In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about setting up an independent research program. He decided to work on inositol-containing phospholipids and was able to synthesize and characterize d-myoinositol 1-phosphate. He also spent several years isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide. This culminated in his discovery of d-myoinositol l,4,5-trisphosphate or IP₃. More information on Ballou''s studies of these phosphoinositides can be found in his JBC Reflections (2).Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at the National Center for Scientific Research (CNRS) in France studying the glycophosphoinositides of mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experiments to establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositol ring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structures of the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4–6). Ballou and his postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intact dimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBC Classics reprinted here.In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyze the biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reported that the major products are a family of three dimannophosphoinositides (A, B, and C) that differ in the number of fatty acyl groups they contain (four, three, and two, respectively). On the basis of their results, they proposed that guanosine diphosphate mannose acts as the sugar donor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside (dimannophosphoinositide C), which is then acylated in a two-step process to first yield dimannophosphoinositide B and then dimannophosphoinositide A.In the second JBC Classic, Ballou and Brennan provide further evidence for their proposed biosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fatty acids into the dimannophosphoinositides. They showed that label from [¹⁴C]palmityl-CoA is incorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After a short incubation period, this molecule is converted to dimannophosphoinositide A, but with longer incubation periods the product is deacylated to isomeric forms of dimannophosphoinositides B and C.Brennan continued to work on these glycophosphoinositides after completing his postdoctoral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) and lipoarabinomannan (LAM)) were multiglycosylated extensions of Ballou''s phosphatidylinositol mannosides and are very important in the pathogenesis of tuberculosis and leprosy. More recent research has defined the biochemistry and genetics of synthesis of these molecules. Brennan is currently University Distinguished Professor in the Department of Microbiology, Immunology, and Pathology at Colorado State University. He served on the editorial board of the Journal of Biological Chemistry for several years and was also named Colorado State University Researcher of the Year in 1992.In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California, Berkeley, although he continued research and teaching for a few years. In recognition of his contributions to science, Ballou has received many awards and honors including election to the National Academy of Sciences (1975), the American Chemical Society''s Claude Hudson Award in Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the University of Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), a National Science Foundation Senior Fellowship (1961), and a University of California Berkeley Citation (1992). Ballou also served as an editorial board member for the Journal of Biological Chemistry.     

The Mechanism of Amino Acid Activation: the Work of Mahlon Hoagland

Enzymatic Carboxyl Activation of Amino Acids(Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. (1956) J. Biol. Chem. 218, 345–358)Mahlon Bush Hoagland was born in Boston, Massachusetts in 1921. He attended Harvard University and graduated in 1943. Knowing that he wanted to be a surgeon, Hoagland then enrolled at Harvard Medical School. However, he was diagnosed with tuberculosis, and his poor health prevented him from becoming a surgeon when he received his M.D. in 1948. Instead, he accepted a research position at Massachusetts General Hospital. In 1953, he became a postdoctoral fellow with Journal of Biological Chemistry (JBC) Classic author Fritz Lipmann (1) at Huntington Laboratories (also at Massachusetts General Hospital), and a year later, he moved to an adjoining laboratory to work on protein synthesis with JBC Classic author Paul Zamecnik (2).Open in a separate windowMahlon HoaglandInspired by Lipmann''s insights into acyl activation mechanisms, Hoagland used a cell-free system created by Zamecnik that carried out net peptide bond formation using ¹⁴C-amino acids (3) to uncover the mechanism of amino acid activation. As reported in the JBC Classic reprinted here, he isolated an enzyme fraction that, in the presence of ATP and amino acids, catalyzed the first step in protein synthesis: the formation of aminoacyl adenylates or activated amino acids. Using data from analysis of this fraction, Hoagland presented a scheme for amino acid activation in his Classic paper.A few years later, Zamecnik and Hoagland discovered a molecule that is essential for protein synthesis: tRNA. This discovery is the subject of the Zamecnik Classic (2).After the discovery of tRNA, Hoagland spent the next year (1957–1958) at Cambridge University''s Cavendish laboratories working with Francis Crick. During that year he traveled to France to visit the Institute Pasteur in Paris. Experiments begun at the Institute would, by 1960, lead to the discovery of messenger RNA (mRNA).When he returned to the United States, Hoagland was appointed associate professor of microbiology at Harvard Medical School. He remained there until 1967 when he accepted a position as professor at Dartmouth Medical School. In 1970, he became the director of the Worcester Foundation for Experimental Biology, a Massachusetts research institute founded by his father. He retired in 1985 and currently lives in Thetford, Vermont.Hoagland has received several awards and honors in recognition of his contributions to science. These include the 1976 Franklin Medal, the 1982 and 1996 Book Awards from the American Medical Writers Association, and membership in the American Academy of Arts and Sciences and the National Academy of Sciences.     

Yasutomi Nishizuka's Discovery of Protein Kinase C

Studies on a Cyclic Nucleotide-independent Protein Kinase and Its Proenzyme in Mammalian Tissues. I. Purification and Characterization of an Active Enzyme from Bovine Cerebellum (Takai, Y., Kishimoto, A., Inoue, M., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7603–7609)Direct Activation of Calcium-activated, Phospholipid-dependent Protein Kinase by Tumor-promoting Phorbol Esters (Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U., and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847–7851)Yasutomi Nishizuka (1932–2004) was born in Ashiya-city, Japan. He attended Kyoto University and obtained his M.D. in 1957 and his Ph.D. in 1962, working with Journal of Biological Chemistry (JBC) Classic author Osamu Hayaishi (1). He then spent a year as a postdoctoral fellow at Rockefeller University with Fritz Lipmann (also featured in a JBC Classic (2)) before returning to Kyoto University to resume work with Hayaishi. During this time, Nishizuka studied the biosynthesis of nicotinamide adenine dinucleotide (NAD), the involvement of GTP in ribosomal protein translation, and ADP-ribosylation by diphtheria toxin.Open in a separate windowYasutomi NishizukaIn 1969, Nishizuka accepted the position of full professor and head of the department of biochemistry at the Kobe University School of Medicine. There, Nishizuka became interested in the role of protein kinases in the regulation of cell functions. This led to his discovery of a novel protein kinase, which he published in the first paper reprinted here as the JBC Classic. As Nishizuka reported in that paper, he and his colleagues partially purified the kinase from bovine cerebellum. They found that the enzyme was capable of phosphorylating histone and protamine and that it probably was produced from its precursor protein by a limited proteolytic reaction. The detailed properties of the proenzyme and its conversion to active protein kinase were reported in a subsequent JBC paper (3). Nishizuka named this new enzyme “protein kinase C (PKC).”A paper published by Nishizuka two years later in the JBC (4) showed that PKC was activated without limited proteolysis by a membrane-associated factor in the presence of a low concentration of Ca²⁺. In 1980, he published another paper in the JBC (5) showing that the membrane-associated factor was diacylglycerol, which suggested that the lipid could be a novel second messenger generated by receptor-stimulated phosphoinositide hydrolysis. Nishizuka validated this idea by showing that treating platelets with a combination of a Ca²⁺ ionophore and membrane-permeant short chain diacylglycerol mimicked stimulation by the aggregating agent thrombin (6). This discovery was a major advance in the understanding of cell signaling.Nishizuka and his colleagues then discovered that PKC is the biological target of tumor-promoting phorbol esters. At that time, it was well known that croton oil augmented carcinogenesis when it was applied at weekly intervals to the skin of mice in conjunction with a very dilute solution of benz[a]pyrene in acetone. The oil contained phorbol ester, a powerful tumor promoter, and caused a wide variety of cellular responses that were similar to those seen with hormones. Nishizuka speculated that the phorbol ester was producing diacylglycerol to activate PKC. However, upon further investigation, he realized that the phorbol ester contained a diacylglycerol-like structure and thus might activate PKC directly. In a series of experiments, published in the second JBC Classic reprinted here, Nishizuka was able to show that the phorbol ester activated PKC directly. This discovery showed that PKC was important for cell proliferation and cancer. It also established the use of phorbol esters as crucial tools for the manipulation of PKC activation in intact cells, eventually allowing the elucidation of the wide range of cellular processes regulated by this enzyme.This research laid the foundation for an enormous number of studies on the complex PKC family, many of them from Nishizuka''s group.In 1975, Nishizuka became president of the University of Kobe, a position that he held until 2001. He received numerous awards and honors for his research, including the Gairdner Foundation International Award (1988), the Alfred P. Sloan Jr. Prize (1988), the Japan Order of Culture (1988), the Albert Lasker Basic Medical Research Award (1989), the Kyoto Prize (1992), the Wolf Prize in Medicine (1995), the Jimenez Diaz Award (1995), and the Schering Prize (1995). He also served as a foreign member and honorary fellow of various academies, including the National Academies of Science, the Royal Society, l''Academie des Sciences, die Deutsche Akademie der Naturforscher Leopoldina, le Real Academia de Ciencias, the Asiatic Society, and the Japan Academy.¹     

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ∼150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the “W” pocket) and the sialic acid-binding site (the “R” pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT)³ produced by the spore-forming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicle-associated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ∼150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (∼50 kDa) and a C-terminal heavy chain domain (∼100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptor-binding domain (HCR/T, ∼50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of α-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5–9). Current models implicate a dual receptor mechanism for the binding of the clostridial neurotoxins to neurons, which includes a ganglioside-binding component (10). Complex gangliosides are sialic acid-containing glycosphingolipids that are located on the outer leaflet of cell membranes and contain a common “core” (GA1) consisting of Gal(β1–3)GalNAc(β1–4)Gal(β1–4)Glc(β1–1)Cer to which one or more N-acetylneuraminic acids (sialic acids) are bound, yielding “a” and “b” series gangliosides (11, 12). Numerous structural and biochemical studies have established that HCR/T contains two carbohydrate-binding sites: a lactose-binding site and a sialic acid-binding site (13). Previous studies showed that Trp¹²⁸⁹ is the key residue for the lactose-binding site, and Arg¹²²⁶ is the key residue for the sialic acid-binding site (14). In this study, we denote the lactose-binding site as the “W” pocket and the sialic acid-binding site as the “R” pocket. Binz and co-workers (14) showed that functional R and W binding sites were required for TeNT toxicity (7). These biochemical and cellular studies were supported by a co-crystal structure of HCR/T bound to a GT1b-β anomer analog, which showed that the W and R carbohydrate-binding pockets were located at different regions of TeNT (7). We recently reported that the W pocket binds gangliosides via the GA1 core structure, whereas the R pocket binds gangliosides via di- or tri-sialic acid moieties (15) where simultaneous binding of TeNT to two gangliosides was synergistic (see Fig. 1a). In the current study, gangliosides were identified that bound exclusively to either the W pocket or R pocket, which allowed the characterization of the role of ganglioside binding to the W and R pockets as dual receptors for TeNT entry into neurons.Open in a separate windowFIGURE 1.Interaction of the HCR domain of TeNT with its putative cellular receptor. a, HCR/T has two ganglioside-binding sites. The W pocket binds to the terminal GalNAc-Gal of the ganglioside (illustrated by GM1a). The R pocket binds to the di-sialic acid of the ganglioside (illustrated by GD3). b, alternating lanes of molecular mass marker proteins and cortical neuron lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was stained for protein with Ponceau S (bottom panel), and then the membrane strips were incubated with 10 nm of the indicated HCR/T (HCR/T wild type (wt), HCR/T (R+, W−), HCR/T (R−, W+), or HCR/T (R−, W−)) followed by HRP-conjugated α-FLAG antibody. The bands were visualized with SuperSignal; exposed film is shown (upper panel). The asterisk denotes the position of purified gangliosides resolved under identical conditions. Migration of the molecular mass marker proteins is indicated (kDa) in the left-most lane in the upper panel.     

Germ Line-governed Recognition of a Cancer Epitope by an Immunodominant Human T-cell Receptor

CD8⁺ T-cells specific for MART-1-(26–35), a dominant melanoma epitope restricted by human leukocyte antigen (HLA)-A*0201, are exceptionally common in the naive T-cell repertoire. Remarkably, the TRAV12-2 gene is used to encode the T-cell receptor α (TCRα) chain in >87% of these T-cells. Here, the molecular basis for this genetic bias is revealed from the structural and thermodynamic properties of an archetypal TRAV12-2-encoded TCR complexed to the clinically relevant heteroclitic peptide, ELAGIGILTV, bound to HLA-A*0201 (A2-ELA). Unusually, the TRAV12-2 germ line-encoded regions of the TCR dominate the major atomic contacts with the peptide at the TCR/A2-ELA interface. This “innate” pattern of antigen recognition probably explains the unique characteristics and extraordinary frequencies of CD8⁺ T-cell responses to this epitope.Malignant melanoma is responsible for 75% of all skin cancer-related deaths worldwide, and the global incidence is rising. The MART-1 (1) protein, also known as Melan-A (2), is expressed by virtually all fresh melanoma tumor specimens and elicits natural CD8⁺ T-cell responses (3, 4) that can lead to spontaneous disease regression (reviewed in Ref. 5). Consequently, CD8⁺ T-cell responses directed against the MART-1 protein have been investigated extensively (reviewed in Refs. 2, 6, and 7), and heteroclitic forms of the dominant MART-1-(26–35) peptide epitope (8, 9), which is restricted by human leukocyte antigen (HLA)-A*0201, are currently being used in a number of clinical trials (10–12). In recent developments, adoptive T-cell therapy directed against the MART-1 protein has been used to mediate cancer regression in ∼50% of late stage melanoma patients (13). However, these approaches have not proved to be universally effective, and there remains considerable scope for improvement. In order to design more effective immune-based therapies against the MART-1 protein, it is essential to understand the precise molecular rules that govern the interaction between T-cell receptors (TCRs)⁶ and the HLA-A*0201·MART-1-(26–35) complex. Previous structural studies of human TCR/peptide major histocompatibility complex (pMHC) interactions (14–16) indicate that specific regions of the TCR have different roles during antigen engagement; thus, the germ line-encoded complementarity-determining region 1 and 2 (CDR1 and -2) loops contact mainly the conserved helical region of the MHC surface, and the more variable somatically rearranged CDR3 loops contact mainly the antigenic peptide. Dissecting the nature of these contacts, which have been shown to be highly variable for individual TCR/pMHC interactions (17–19), is an important step toward understanding the principles of antigen recognition and for the development of improved T-cell vaccines (20). However, the current data base of human TCR·pMHC complexes reported in the literature is limited (∼16), compared with >100 antibody-antigen structures. This has made it difficult to ascertain whether there are conserved binding modes for TCR/pMHC interactions dictated by a number of specific contacts or whether there are potentially unlimited numbers of TCR docking orientations dependent on the nature of individual recognition events. Furthermore, there are no examples to date of human TCR·pMHC class I structures in which the bound peptide is a decamer; this represents a substantial deficiency in our current knowledge, given the preponderance with which decamer peptides are processed, presented, and recognized. The low number of TCR·pMHC complex structures solved to date reflects technical difficulties inherent in the production of soluble TCR and pMHC molecules that retain stability and challenges related to the crystallization of complexes with relatively low binding affinities (K_D = 0.1–500 μm) (21, 22). In general, TCRs specific for tumor-derived epitopes bind in the weaker range of TCR/pMHC affinities (21). This obstacle to the generation of high quality co-complex crystals is underscored by the fact that only one other tumor-specific human TCR·pMHCI complex structure has been documented previously (23).In this study, we expressed a soluble TCR (MEL5) specific for ELAGIGILTV, the common MART-1-(26–35) heteroclitic peptide, complexed to HLA-A*0201 (A2-ELA). Notably, HLA-A*0201 is the most common HLA allele in the human population (24). The CDR1 and CDR2 loops of this TCR are encoded by the TRAV12-2 and TRBV30 genes (International Immunogenetics (IMGT) nomenclature). Interestingly, the TRAV12-2 gene is expressed in the vast majority of CD8⁺ T-cell populations specific for HLA-A*0201·MART-1-(26–35) across multiple individuals (25, 26). To resolve the enigma of the dominant TRAV12-2 gene and determine the molecular characteristics that govern CD8⁺ T-cell recognition of the HLA-A*0201·MART-1-(26–35) antigen, we performed a biophysical, thermodynamic, and structural analysis of MEL5 TCR binding to A2-ELA. The data provide a molecular basis for biased TCR gene product selection in the CD8⁺ T-cell response to HLA-A*0201·MART-1-(26–35) and indicate that pMHC antigens can be subject to “innate-like” binding modes within adaptive immune responses.     

Myosin II Recruitment during Cytokinesis Independent of Centralspindlin-mediated Phosphorylation

During cell division, the mechanisms by which myosin II is recruited to the contractile ring are not fully understood. Much recent work has focused on a model in which spatially restricted de novo filament assembly occurs at the cell equator via localized myosin II regulatory light chain (RLC) phosphorylation, stimulated by the RhoA-activating centralspindlin complex. Here, we show that a recombinant myosin IIA protein that assembles constitutively and is incapable of binding RLC still displays strong localization to the furrow in mammalian cells. Furthermore, this RLC-deficient myosin II efficiently drives cytokinesis, demonstrating that centralspindlin-based RLC phosphorylation is not necessary for myosin II localization during furrowing. Myosin II truncation analysis further reveals two distinct myosin II tail properties that contribute to furrow localization: a central tail domain mediating cortical furrow binding to heterologous binding partners and a carboxyl-terminal region mediating co-assembly with existing furrow myosin IIA or IIB filaments.Non-muscle myosin II, through its interaction with F-actin, is believed to be the dominant force-producing machinery utilized to separate daughter cells during cell division. Following anaphase onset, myosin II is recruited to the equatorial cortex where it assembles into the contractile ring. Despite much recent progress, the exact mechanism by which myosin II is recruited to and retained in the contractile ring in the proper spatio-temporal manner remains unclear.Myosin II is a member of the myosin superfamily that binds F-actin and hydrolyzes ATP to produce force. A monomer consists of two myosin heavy chains (“MHCs”),³ two essential light chains (“ELCs”), and two regulatory light chains (“RLCs”) (see Fig. 1A). The MHC consists of an amino-terminal globular head domain often referred to as the “motor” domain. It is responsible for F-actin binding and ATP binding and hydrolysis. One RLC and one ELC associate with each MHC via two IQ motifs on a neck region linking the head and tail domain. The remainder of the MHC forms a continuous α-helix that interacts with another MHC rod to create a coiled-coil-mediated dimer. At the extreme C terminus, mammalian non-muscle myosin II molecules contain an ∼30 residue “non-helical tailpiece.” Many phosphorylation sites have been identified on both the RLC and the MHC (1–4). The best characterized of these sites is Thr-18/Ser-19 on the RLC, which, when phosphorylated, has been shown to activate myosin II by increasing the affinity of MHC for F-actin, consequently increasing the ATPase activity (5, 6). Phosphorylation of these sites is also able to convert myosin II from a folded 10 S “inactive” monomer into the extended 6 S monomer that readily forms filaments (7).Open in a separate windowFIGURE 1.RLC-independent localization of myosin to furrow in HeLa and COS-7 cells. A, diagram of myosin IIA. GFP was conjugated to the amino terminus of the MHC. B, diagram of GFP-IIA constructs. GFP-IIA-ΔIQ2 removes the RLC binding site known as the IQ2 motif. C and D, at 72 h after transfection, HeLa (C) or COS-7 (D) cells expressing GFP-IIA (top row) or GFP-IIA-ΔIQ2 in early anaphase (middle row) or late anaphase (lower row) were fixed and stained with phalloidin-568 (red) for actin and DAPI (blue) for DNA. The images in the right column are merges of actin, DNA, and GFP channels.Mammalian genomes contain three genes encoding non-muscle myosin II heavy chain isoforms, mhc IIA, IIB, and IIC. MHC IIA and IIB are widely expressed in many tissues and cell lines, whereas IIC is expressed with a more limited distribution (8). In mice, gene knockouts of mhc IIA and IIB result in differing phenotypes that are only partially rescued by the other isoform, suggesting that both isoform-specific and overlapping roles exist (9). Previous reports have suggested that myosin IIA and IIB isoforms are capable of co-assembling into mixed or heterotypic filaments (10, 11). However, there is also evidence showing that myosin II isoforms have different subcellular localization in non-mitotic cells, supportinga model in which homotypic filaments are the dominant filamentous structure in live cells (12, 13). Whether myosin IIA and IIB can co-assemble in the contractile ring of dividing cells is not known.The dominant model for furrow localization of myosin II during cell division hypothesizes spatially restricted equatorial activation and filament assembly via phosphorylation of the RLC on Thr-18/Ser-19. The most prominent upstream signaling pathway implicated in this furrow recruitment model is the centralspindlin pathway. In this pathway, the kinesin-6 protein MKLP1 anchors MgcRacGAP and a RhoGEF (ECT2) to the spindle midzone (14). This in turn locally activates RhoA, leading to activation of Rho kinase and/or citron kinase, both of which have been shown capable of phosphorylating RLC (15–18). Centralspindlin-based signals clearly contribute to myosin II-cytokinesis functions. However, whether these signals contribute to initial myosin II binding/recruitment, to myosin II contractile activation, or to both, is unresolved.Another recent study reported that GFP-tagged RLC constructs with alanine substitutions at the activating Thr-18/Ser-19 sites were still able to localize to the furrow of dividing HeLa cells, suggesting that RLC phosphorylation is not required for myosin equatorial localization (19). However, in that study, it was not clear how much endogenous wild type RLC was present; thus this result may represent a tracer amount of the T18A/S19A mutant RLC passively co-assembling with a larger pool of endogenous RLC-phosphorylated myosin II.Another proposed model for myosin recruitment to the equatorial region of a dividing cell is cortical flow. In this model, supported by observations in both Dictyostelium and mammalian cells, myosin filaments move along the cortex and into the furrow in a motor-dependent manner (20–22). However, recent studies using total internal reflection fluorescence imaging in normal rat kidney cells revealed no detectable myosin II cortical flow (23), raising uncertainty as to whether cortical flow is an important mechanism for myosin recruitment in mammalian cells.In this study, we provide the first evidence that mammalian myosin II can localize to the furrow of dividing cells independent of the regulatory light chain. These studies demonstrate that both robust myosin II recruitment to the furrow and efficient cell division can be achieved without spatially localized centralspindlin-mediated RLC phosphorylation. We conclude that other mechanisms such as cortical flow (22, 24) and/or equatorial myosin II binding partners (25, 26) must be sufficient for myosin II recruitment and cell furrowing in mammalian cells. Furthermore, we show that a headless myosin construct can localize to the contractile ring, supporting a model in which actin binding and ATPase activity are not required for myosin II recruitment. We also provide novel evidence that MHC isoforms are capable of co-assembling in the contractile ring.     

Structure-Function Relationships in Ribonuclease S: the Work of Frederic M. Richards

The Preparation of Subtilisin-modified Ribonuclease and the Separation of the Peptide and Protein Components(Richards, F. M., and Vithayathil, P. (1959) J. Biol. Chem. 234, 1459–1465)The Three-dimensional Structure of Ribonuclease-S. Interpretation of an Electron Density Map at a Nominal Resolution of 2 Å(Wyckoff, H. W., Tsernoglou, D., Hanson, A. W., Knox, J. R., Lee, B., and Richards, F. M. (1970) J. Biol. Chem. 245, 305–328)Frederic Middlebrook Richards (1925–2009) was born in New York City. He attended the Massachusetts Institute of Technology and, after a brief stint in the military, received his B.S. in 1948. Richards then enrolled in graduate school at Harvard Medical School, where he worked with Barbara Low and received his Ph.D. in 1952. After graduating he remained at Harvard for another year as a research fellow with Edwin Joseph Cohn, who was featured in a previous Journal of Biological Chemistry (JBC) Classic (1). Richards then moved to the Carlsberg Laboratory in Denmark where, with Kaj Linderstrøm-Lang and others, he began working on ribonuclease.Open in a separate windowFrederic M. RichardsAfter a short stint as a postdoctoral fellow at Cambridge University, Richards joined the faculty of the Department of Biochemistry at Yale University in 1955 as an assistant professor. He rose rapidly through the ranks, becoming professor in 1963. That year, Richards was also appointed chairman of the Department of Molecular Biology and Biophysics at Yale, which entailed a move from the Medical School to the Yale College campus. Following a sabbatical at Oxford University in 1967–1968, for which Richards and his wife Sally sailed their own boat with a small crew across the Atlantic Ocean, Yale merged the Medical School Department of Biochemistry and the Yale College Department of Molecular Biology and Biophysics to form a new university-wide Department of Molecular Biophysics & Biochemistry (MB&B) with Richards as its founding chair (1969–1973). Richards remained at Yale for his entire research career, eventually becoming Sterling Professor of Molecular Biophysics and Biochemistry.Much of Richard''s early research centered on bovine pancreatic ribonuclease (RNase). During his time at the Carlsberg laboratory, he showed that cleavage of RNase by the protease subtilisin produces a modified RNase (RNase S) that is still active (2). After starting his own lab at Yale, Richards was able to separate RNase S into a 20-residue S-peptide and a 102-residue S-protein, both of which lacked enzymatic activity. However, when the peptide and protein were recombined, the activity was recovered. Richards published an initial paper on this finding in 1958 (3). He followed this up with a more extensive article in the JBC, which is reprinted here as the first JBC Classic. In this paper, Richards and co-workers purified and characterized RNase S, separated it into S-peptide and S-protein, showed that almost all enzymatic activity is recovered when the two components are recombined, and also reported that the only observed change in covalent structure during the conversion of RNase A to RNase S is the hydrolysis of the peptide bond between residues 20 and 21.The demonstration that two separate, inactive fragments of the enzyme RNase A could be reconstituted to form an active enzyme provided the first experimental evidence that the ability of a protein to form a three-dimensional structure is an intrinsic property of its amino acid sequence. This work also foreshadowed the extensive RNase A refolding studies performed by Nobel laureate Christian Anfinsen, as discussed in a previous JBC Classic (4).In the 1960s Richards teamed up with Harold Wyckoff to solve the three-dimensional structure of RNase S. Initially, in 1967, they produced a 3.5 Å electron density map (5), which they used to determine the approximate conformation of the peptide chain. Three years later, they collected data to 2 Å, as reported in the second JBC Classic reprinted here. Using these data, Richards, Wyckoff, and colleagues produced an electron density map, which they used to determine the complete three-dimensional structure of RNase S. This structure tied with three others for the third protein structure ever solved to atomic resolution. Richards also showed that RNase S was enzymatically active in crystal form, putting to rest the widely held view at that time that protein crystal structures were irrelevant to the conformation and behavior of enzymes in solution.Richards received many honors and awards for his scientific achievements, including the Pfizer-Paul Lewis Award in Enzyme Chemistry (1965), election as Fellow of the American Academy of Arts and Sciences (1968), election to the National Academy of Sciences (1971), the Kai Linderstrøm-Lang Prize in Protein Chemistry (1978), the American Society for Biochemistry and Molecular Biology Merck Award (1988), the Stein and Moore Award of the Protein Society (1988), and the State of Connecticut Medal of Science (1995). He was also president of ASBMB (1979) and the Biophysical Society (1972–1973).