Crystal structure of a hypothetical protein,TM841 of Thermotoga maritima,reveals its function as a fatty acid-binding protein

We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution. The protein belongs to a large protein family, DegV or COG1307 of unknown function. The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures. These structural homologies, however, provided no clues for the function of TM841. But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains. The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism.     

Crystal complexes of a predicted S-adenosylmethionine-dependent methyltransferase reveal a typical AdoMet binding domain and a substrate recognition domain

S-adenosyl-L-methionine-dependent methyltransferases (MTs) are abundant, and highly conserved across phylogeny. These enzymes use the cofactor AdoMet to methylate a wide variety of molecular targets, thereby modulating important cellular and metabolic activities. Thermotoga maritima protein 0872 (TM0872) belongs to a large sequence family of predicted MTs, ranging phylogenetically from relatively simple bacteria to humans. The genes for many of the bacterial homologs are located within operons involved in cell wall synthesis and cell division. Despite preliminary biochemical studies in E. coli and B. subtilis, the substrate specificity of this group of more than 150 proteins is unknown. As part of the Midwest Center for Structural Genomics initiative (www.mcsg.anl.gov), we have determined the structure of TM0872 in complexes with AdoMet and with S-adenosyl-L-homocysteine (AdoHcy). As predicted, TM0872 has a typical MT domain, and binds endogenous AdoMet, or co-crystallized AdoHcy, in a manner consistent with other known MT structures. In addition, TM0872 has a second domain that is novel among MTs in both its location in the sequence and its structure. The second domain likely acts in substrate recognition and binding, and there is a potential substrate-binding cleft spanning the two domains. This long and narrow cleft is lined with positively charged residues which are located opposite the S(+)-CH(3) bond, suggesting that a negatively charged molecule might be targeted for catalysis. However, AdoMet and AdoHcy are both buried, and access to the methyl group would presumably require structural rearrangement. These TM0872 crystal structures offer the first structural glimpses at this phylogenetically conserved sequence family.     

Transmembrane helices before, during, and after insertion

The transmembrane (TM) helix is the fundamental structural unit of helix-bundle membrane proteins. Recent biophysical studies provide new insights into the interactions of TM helices with each other and with membrane lipid bilayers. The biological process of helix insertion is carried out by translocon complexes acting in concert with ribosomes. An electron cryo-microscopic reconstruction of these complexes reveals their architecture in new detail, and shows that the complex is constructed from four SecY/Sec61 heterotrimers and two TRAP complexes. A disulfide bridge study shows that elongating polypeptide chains pass through the pore previously identified in the X-ray structure of an archaeal SecY heterotrimer. The fundamental code used by the translocon to select polypeptide segments for insertion as TM helices has been broken. A detailed analysis of the TM amino acid distributions of helix-bundle membrane proteins of known structure recapitulates this code.     

Structural and functional analysis of the Na+/H+ exchanger

The mammalian NHE (Na+/H+ exchanger) is a ubiquitously expressed integral membrane protein that regulates intracellular pH by removing a proton in exchange for an extracellular sodium ion. Of the nine known isoforms of the mammalian NHEs, the first isoform discovered (NHE1) is the most thoroughly characterized. NHE1 is involved in numerous physiological processes in mammals, including regulation of intracellular pH, cell-volume control, cytoskeletal organization, heart disease and cancer. NHE comprises two domains: an N-terminal membrane domain that functions to transport ions, and a C-terminal cytoplasmic regulatory domain that regulates the activity and mediates cytoskeletal interactions. Although the exact mechanism of transport by NHE1 remains elusive, recent studies have identified amino acid residues that are important for NHE function. In addition, progress has been made regarding the elucidation of the structure of NHEs. Specifically, the structure of a single TM (transmembrane) segment from NHE1 has been solved, and the high-resolution structure of the bacterial Na+/H+ antiporter NhaA has recently been elucidated. In this review we discuss what is known about both functional and structural aspects of NHE1. We relate the known structural data for NHE1 to the NhaA structure, where TM IV of NHE1 shows surprising structural similarity with TM IV of NhaA, despite little primary sequence similarity. Further experiments that will be required to fully understand the mechanism of transport and regulation of the NHE1 protein are discussed.     

Hypothetical protein AF2241 from Archaeoglobus fulgidus adopts a cyclophilin-like fold

AF2241 is a hypothetical protein from Archaeoglobus fulgidus and it belongs to the PFam domain of unknown function 369 (DUF369). NMR structural determination reveals that AF2241 adopts a cyclophilin-like fold, with a β-barrel core composed of eight β-strands, one α-helix, and one 3₁₀ helix located at each end of the barrel. The protein displays a high structural similarity to TM1367, another member of DUF369 whose structure has been determined recently by X-ray crystallography. Structural similarity search shows that AF2241 also has a high similarity to human cyclophilin A, however, sequence alignment and electrostatic potential analysis reveal that the residues in the PPIase catalytic site of human cyclophilin A are not conserved in AF2241 or TM1367. Instead, a putative active site of AF2241 maps to a negatively charged pocket composed of 9 conserved residues. Our results suggest that although AF2241 adopts the same fold as the human cyclophilin A, it may have distinct biological function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Predicted role for the archease protein family based on structural and sequence analysis of TM1083 and MTH1598, two proteins structurally characterized through structural genomics efforts

Recently, the structures of two proteins belonging to the archease family, TM1083 from Thermotoga maritima and MTH1598 from Methanobacterium thermoautotrophicum, have been solved independently by two Protein Structure Initiative structural genomics pilot centers using X-ray crystallography and NMR, respectively. The archease protein family is a good example of one of the paradoxes of structural genomics: Approximately one third of protein structures produced by structural genomics centers have no known function and are still annotated as "hypothetical proteins" in the Protein Data Bank. In the case of archeases, despite the existence of two protein structures and abundant sequence information, there is still no function assigned to this protein family. Here, our group predicts, based on structural similarity, sequence conservation, and gene context analyses, that members of this protein family might function as chaperones or modulators of proteins involved in DNA/RNA processing. The conservation of genomic context for this protein family is constant from Archaea and Bacteria to humans, and suggests that unannotated open reading frames contiguous to them could be novel RNA/DNA binding proteins.     

Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.     

Crystal structure of ST2348, a CBS domain protein, from hyperthermophilic archaeon Sulfolobus tokodaii

The crystal structure of a hypothetical protein ST2348 (GI: 47118305) from the hyperthermophilic bacteria Sulfolobus tokodaii has been determined using X-ray crystallography. The protein consists of two CBS (cystathione β synthase) domains, whose function has been analyzed and reported here. PSI-BLAST shows a conservation of this domain in about 100 proteins in various species. However, none of the close homologs of ST2348 have been functionally characterized so far. Structure and sequence comparison of ST2348 with human AMP-kinase γ1 subunit and the CBS domain pair of bacterial IMP dehydrogenase is suggestive of its binding to AMP and ATP. A highly conserved residue Asp118, located in a negatively charged patch near the ligand binding cleft, could serve as a site for phosphorylation similar to that found in the chemotatic signal protein CheY and thereby ST2348 can function as a signal transduction molecule.     

19F NMR studies of the D-galactose chemosensory receptor. 1. Sugar binding yields a global structural change.

The Escherichia coli D-galactose and D-glucose receptor is an aqueous sugar-binding protein and the first component in the distinct chemosensory and transport pathways for these sugars. Activation of the receptor occurs when the sugar binds and induces a conformational change, which in turn enables docking to specific membrane proteins. Only the structure of the activated receptor containing bound D-glucose is known. To investigate the sugar-induced structural change, we have used 19F NMR to probe 12 sites widely distributed in the receptor molecule. Five sites are tryptophan positions probed by incorporation of 5-fluorotryptophan; the resulting 19F NMR resonances were assigned by site-directed mutagenesis. The other seven sites are phenylalanine positions probed by incorporation of 3-fluorophenylalanine. Sugar binding to the substrate binding cleft was observed to trigger a global structural change detected via 19F NMR frequency shifts at 10 of the 12 labeled sites. Two of the altered sites lie in the substrate binding cleft in van der Waals contact with the bound sugar molecule. The other eight altered sites, specifically two tryptophans and six phenylalanines distributed equally between the two receptor domains, are distant from the cleft and therefore experience allosteric structural changes upon sugar binding. The results are consistent with a model in which multiple secondary structural elements, known to extend between the substrate cleft and the protein surface, undergo shifts in their average positions upon sugar binding to the cleft. Such structural coupling provides a mechanism by which sugar binding to the substrate cleft can cause structural changes at one or more docking sites on the receptor surface.     

Crystal structure of the conserved protein TTHA0727 from Thermus thermophilus HB8 at 1.9 A resolution: A CMD family member distinct from carboxymuconolactone decarboxylase (CMD) and AhpD

TTHA0727 is a conserved hypothetical protein from Thermus thermophilus HB8, with a molecular mass of 12.6 kDa. TTHA0727 belongs to the carboxymuconolactone decarboxylase (CMD) family (Pfam 02627). A sequence comparison with its homologs suggested that TTHA0727 is a distinct protein from alkylhydroperoxidase AhpD and gamma-carboxymuconolactone decarboxylase in the CMD family. Here we report the 1.9 A crystal structure of TTHA0727 (PDB ID: 2CWQ) determined by the multiwavelength anomalous dispersion method. The TTHA0727 monomer structure consists of seven alpha-helices (alpha1-alpha7) and one short 3(10)-helix. The crystal structure and the analytical ultracentrifugation revealed that TTHA0727 forms a hexameric ring structure in solution. The electrostatic potential distribution on the solvent-accessible surface of the TTHA0727 hexamer showed that positively charged regions exist on the side of the ring structure, suggesting that TTHA0727 interacts with some negatively charged molecules. A structural homology search revealed that the structure of three alpha-helices (alpha4-alpha6) is remarkably conserved, suggesting that it is the common structural motif for the CMD family proteins. In addition, the nine residues of the N-terminal tag bound to the cleft region between alpha1 and alpha3 in chains A and B of TTHA0727, implying that this region is the putative binding/active site for some small molecules.     

Structure of Bacillus subtilis YXKO--a member of the UPF0031 family and a putative kinase

We determined the 1.6-A resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY-CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus. YXKO homologues are broadly distributed and are by and large described as proteins with unknown function. The YXKO protein has an alpha/beta fold and shows high structural homology to the members of a ribokinase-like superfamily. However, YXKO is the only member of this superfamily known to form tetramers. Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg(2+)-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily. Two adjacent monomers contribute residues to the active site. The crystal structure provides valuable information about the YXKO protein's tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.     

The first structure of an RNA m5C methyltransferase, Fmu, provides insight into catalytic mechanism and specific binding of RNA substrate

The crystal structure of E. coli Fmu, determined at 1.65 A resolution for the apoenzyme and 2.1 A resolution in complex with AdoMet, is the first representative of the 5-methylcytosine RNA methyltransferase family that includes the human nucleolar proliferation-associated protein p120. Fmu contains three subdomains which share structural homology to DNA m(5)C methyltransferases and two RNA binding protein families. In the binary complex, the AdoMet cofactor is positioned within the active site near a novel arrangement of two conserved cysteines that function in cytosine methylation. The site is surrounded by a positively charged cleft large enough to bind its unique target stem loop within 16S rRNA. Docking of this stem loop RNA into the structure followed by molecular mechanics shows that the Fmu structure is consistent with binding to the folded RNA substrate.     

Crystal structure of the autocatalytic initiator of glycogen biosynthesis,glycogenin

The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.     

Synthesis, activity, and preliminary structure of the fourth EGF-like domain of thrombomodulin.

The fourth EGF-like domain of thrombomodulin (TM4), residues E346-F389 in the TM sequence, has been synthesized. Refolding of the synthetic product under redox conditions gave a single major product. The disulfide bonding pattern of the folded, oxidized domain was (1-3, 2-4, 5-6), which is the same as that found in EGF protein. TM4 was tested for TM anticoagulant activity because deletion and substitution mutagenesis experiments have shown that the fourth EGF-like domain of TM is essential for TM cofactor activity. TM4 showed no TM-like activity in two assay systems, both for inhibition of fibrin clot formation, and for cofactor activity in thrombin activation of protein C. A preliminary structure of TM4 was determined by 2D 1H NMR from 519 NOE-derived distance constraints. Distance geometry calculations yielded a single convergent structure. The structure resembles the structure of EGF and other known EGF-like domains but has some key differences. The central two-stranded beta-sheet is conserved despite the differences in the number of amino acids in the loops. The C-terminal loop formed by the disulfide bond between C372 and C386 in TM4 is five amino acids longer than the analogous loop between C33 and C42 of EGF protein. This loop appears to have a different fold in TM4 than in EGF protein. The loop forms the two outside strands of a broken, irregular tri-stranded beta-sheet, and amino acids H384-F389 lie between the two strands forming the middle strand of the sheet. Thus, although the C-terminus of EGF protein forms one of the outside strands of a tri-stranded antiparallel sheet, the C-terminus of TM4 forms the inside strand of an irregular tri-stranded parallel-anti-parallel sheet. The residues D349, E357, and E374, which were shown to be critical for cofactor activity by alanine scanning mutagenesis, all lie in a patch near the C-terminal loop, and are solvent accessible. The other critical residues, Y358 and F376, are largely buried and appear to play essential structural rather than functional roles.     

The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease

Alphavirus replication and propagation is dependent on the protease activity of the viral nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into functional components. Thus, nsP2 is an attractive target for drug discovery efforts to combat highly pathogenic alphaviruses. Unfortunately, antiviral development has been hampered by a lack of structural information for the nsP2 protease. Here, we report the crystal structure of the nsP2 protease (nsP2pro) from Venezuelan equine encephalitis alphavirus determined at 2.45 A resolution. The protease structure consists of two distinct domains. The nsP2pro N-terminal domain contains the catalytic dyad cysteine and histidine residues organized in a protein fold that differs significantly from any known cysteine protease or protein folds. The nsP2pro C-terminal domain displays structural similarity to S-adenosyl-L-methionine-dependent RNA methyltransferases and provides essential elements that contribute to substrate recognition and may also regulate the structure of the substrate binding cleft.     

Crystal structure of a putative CN hydrolase from yeast

The crystal structure of a yeast hypothetical protein with sequence similarity to CN hydrolases has been determined to 2.4 A resolution by the multiwavelength anomalous dispersion (MAD) method. The protein folds as a four-layer alphabetabetaalpha sandwich and exists as a dimer in the crystal and in solution. It was selected in a structural genomics project as representative of CN hydrolases at a time when no structures had been determined for members of this family. Structures for two other members of the family have since been reported and the three proteins have similar topology and dimerization modes, which are distinct from those of other alphabetabetaalpha proteins whose structures are known. The dimers form an unusual eight-layer alphabetabetaalpha:alphabetabetaalpha structure. Although the precise enzymatic reactions catalyzed by the yeast protein are not known, considerable information about the active site may be deduced from conserved sequence motifs, comparative biochemical information, and comparison with known structures of hydrolase active sites. As with serine hydrolases, the active-site nucleophile (cysteine in this case) is positioned on a nucleophile elbow.     

Crystal structures of apo- and holo-bovine alpha-lactalbumin at 2. 2-A resolution reveal an effect of calcium on inter-lobe interactions

High affinity binding of Ca(2+) to alpha-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca(2+) as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca(2+). To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-A resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of alpha-helical and beta-subdomains separated by a cleft. Even though alternative cations and Ca(2+) liganding solvent molecules are absent, removal of Ca(2+) has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr(103) of the helical lobe and Gln(54) of the beta-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca(2+)-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr(103) by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the beta-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca(2+) is mediated by an effect on interactions between the two subdomains.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.