UV difference spectroscopy of ligand binding to maltose-binding protein.

We have used UV absorbance spectroscopy to study the binding of linear and circular maltodextrins to maltose-binding protein (MBP). Titrations with maltose yield three isosbestic points in the difference spectrum of MBP, consistent with two protein conformations: ligand-free and ligand-bound. In contrast, titrations with maltotetraose reveal three conformations: ligand-free, a low-affinity liganded state, and a high affinity liganded state. These results confirm and extend the results from tritium NMR spectroscopy, namely, that MBP can bind maltodextrin either by the sugar's anomeric end (high affinity) or by the middle of the maltodextrin chain (low affinity).     

Dynamical persistence of active sites identified in maltose-binding protein

This study identifies dynamical properties of maltose-binding protein (MBP) useful in unveiling active site residues susceptible to ligand binding. The described methodology has been previously used in support of novel topological techniques of persistent homology and statistical inference in complex, multi-scale, high-dimensional data often encountered in computational biophysics. Here we outline a computational protocol that is based on the anisotropic elastic network models of 14 all-atom three-dimensional protein structures. We introduce the notion of dynamical distance matrices as a measure of correlated interactions among 370 amino acid residues that constitute a single protein. The dynamical distance matrices serve as an input for a persistent homology suite of codes to further distinguish a small subset of residues with high affinity for ligand binding and allosteric activity. In addition, we show that ligand-free closed MBP structures require lower deformation energies than open MBP structures, which may be used in categorization of time-evolving molecular dynamics structures. Analysis of the most probable allosteric coupling pathways between active site residues and the protein exterior is also presented.     

Effects of ligand binding on the internal dynamics of maltose-binding protein.

Ligand binding to proteins often causes large conformational changes. A typical example is maltose-binding protein (MBP), a member of the family of periplasmic binding proteins of Gram-negative bacteria. Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e. the distance between its two domains decreases. In contrast, binding of the larger, nonphysiological ligand beta-cyclodextrin does not result in closure of the binding cleft. We have investigated the dynamic properties of MBP in its different states using time-resolved tryptophan fluorescence anisotropy. We found that the 'empty' protein exhibits strong internal fluctuations that almost vanish upon ligand binding. The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side-chains relative to the protein backbone, and orientational fluctuations of entire domains. After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side-chains become rather restricted. This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger beta-cyclodextrin. The fluctuations of tryptophan side-chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.     

Tritium NMR spectroscopy of ligand binding to maltose-binding protein

Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity. At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30). The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange. We interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.     

Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1

RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.     

Programming xenon diffusion in maltose-binding protein

Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge ～9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST ¹²⁹Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by ¹³C NMR spectroscopy and variable-B₁ hyper-CEST experiments. “Xe flooding” molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP’s large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.     

Subsites of trypsin active site favor catalysis or substrate binding.

Enzymes enhance chemical reaction rates by lowering the activation energy, the energy barrier of the reaction leading to products. This occurs because enzymes bind the high-energy intermediate of the reaction (the transition state) more strongly than the substrate. We studied details of this process by determining the substrate binding energy (DeltaG(s), calculated from K(m) values) and the activation energy (DeltaG(T), determined from k(cat)/K(m) values) for the trypsin-catalyzed hydrolysis of oligopeptides. Plots of DeltaG(T) versus DeltaG(s) for oligopeptides with 15 amino acid replacements at each of the positions P(1)', P(1), and P(2) were straight lines, as predicted by a derived equation that relates DeltaG(T) and DeltaG(s). The data led to the conclusion that the trypsin active site has subsites that bind moieties of substrate and of transition state in characteristic ratios, whichever substrate is used. This was unexpected and means that each subsite characteristically favors substrate binding or catalysis.     

Androgen binding protein is tissue-specifically expressed and biologically active in transgenic mice

In view of the inconclusive data concerning the role of androgen-binding protein (ABP) in male reproductive physiology, we thought it would be pertinent to make several transgenic mouse lines overexpressing the rat ABP gene to unravel its role in Sertoli cell and epididymal homeostasis. Heterozygote transgenic mouse lines carrying the 5.5 kb ABP rat genomic DNA were produced by pronuclear microinjection. Northern blot analysis showed overexpression of rat ABP (rABP) mRNA in the testis of transgenic mice compared to rat testis control. rABP was appropriately expressed in Sertoli cells as demonstrated by in situ hybridization analysis. Sertoli cell number is increased in the seminiferous tubules of mice overexpressing rABP compared to non-transgenic littermates and scattered Sertoli cells present vacuolated-like cytoplasms, PAS and osmium negative. Compared to the wild type, the transgenic mice exhibited reduced fertility and focal damage in seminiferous epithelium characterized by morphological features compatible with programmed cell death.     

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.     

Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase.

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA'), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.     

Demonstration in vivo that interaction of maltose-binding protein with SecB is determined by a kinetic partitioning.

An early step in the export of maltose-binding protein to the periplasm is interaction with the molecular chaperone SecB. We demonstrate that binding to SecB in vivo is determined by a kinetic partitioning between the folding of maltose-binding protein to its native state and its association with SecB. A complex of SecB and a species of maltose-binding protein that folds slowly is shown to be longer-lived than a complex of the wild-type maltose-binding protein and SecB. In addition, we show that incomplete nascent chains, which are unable to fold, remain complexed with SecB.     

Electrostatic environment at the active site of prolyl oligopeptidase is highly influential during substrate binding

The positive electrostatic environment of the active site of prolyl oligopeptidase was investigated by using substrates with glutamic acid at positions P2, P3, P4, and P5, respectively. The different substrates gave various pH rate profiles. The pKa values extracted from the curves are apparent parameters, presumably affected by the nearby charged residues, and do not reflect the ionization of a simple catalytic histidine as found in the classic serine peptidases like chymotrypsin and subtilisin. The temperature dependence of kcat/Km did not produce linear Arrhenius plots, indicating different changes in the individual rate constants with the increase in temperature. This rendered it possible to calculate these constants, i.e. the formation (k1) and decomposition (k-1) of the enzyme-substrate complex and the acylation constant (k2), as well as the corresponding activation energies. The results have revealed the relationship between the complex Michaelis parameters and the individual rate constants. Structure determination of the enzyme-substrate complexes has shown that the different substrates display a uniform binding mode. None of the glutamic acids interacts with a charged group. We conclude that the specific rate constant is controlled by k1 rather than k2 and that the charged residues from the substrate and the enzyme can markedly affect the formation but not the structure of the enzyme-substrate complexes.     

A salt-bridge motif involved in ligand binding and large-scale domain motions of the maltose-binding protein

The uptake of nutrients is essential for the survival of bacterial cells. Many specialized systems have evolved, such as the maltose-dependent ABC transport system that transfers oligosaccharides through the cytoplasmic membrane. The maltose/maltodextrin-binding protein (MBP) serves as an initial high-affinity binding component in the periplasm that delivers the bound sugar into the cognate ABC transporter MalFGK(2). We have investigated the domain motions induced by the binding of the ligand maltotriose into the binding cleft using molecular dynamics simulations. We find that MBP is predominantly in the open state without ligand and in the closed state with ligand bound. Oligosaccharide binding induces a closure motion (30.0 degrees rotation), whereas ligand removal leads to domain opening (32.6 degrees rotation) around a well-defined hinge affecting key areas relevant for chemotaxis and transport. Our simulations suggest that a "hook-and-eye" motif is involved in the binding. A salt bridge between Glu-111 and Lys-15 forms that effectively locks the protein-ligand complex in a semiclosed conformation inhibiting any further opening and promoting complete closure. This previously unrecognized feature seems to secure the ligand in the binding site and keeps MBP in the closed conformation and suggests a role in the initial steps of substrate transport.     

Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.     

Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts

The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.     

Soluble expression of a functionally active Plasmodium falciparum falcipain-2 fused to maltose-binding protein in Escherichia coli

Falcipain-2 (fp2) is a hemoglobinase required for supplying peptides and amino acids for the proliferation of Plasmodium falciparum in blood. The prospect of circumventing its activity thereby serves as a potential strategy for mining drugs for anti-malarial therapy. However, to date, efforts to express soluble and active fp2 in Escherichia coli have been futile. To overcome this problem, fp2 was expressed under an array of conditions including the exploitation of multiple gene constructs in eukaryotic and prokaryotic hosts. A series of experiments led to the finding that the placement of maltose-binding protein (MBP) before the fp2 mature domain was best in availing the soluble expression of the protease. The results also indicate that the prodomain impaired the bacterial expression of the protease and the amino acid residues at the N-terminal segment of mature fp2 can have a significant effect on the folding and solubility of the enzyme. The overexpressed MBP-fp2 fusion protein was purified and shown to be functionally active, providing a very useful alternative to the use of resolubilized enzyme for future study of structure and function of fp2.     

Tertiary structure-dependence of misfolding substitutions in loops of the maltose-binding protein.

We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alphaI/betaB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alphabeta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super-secondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.     

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.     

Escherichia coli primase zinc is sensitive to substrate and cofactor binding.

The ligation state of the single zinc site in primase from Escherichia coli changes when various substrates and cofactors are added alone or in combination as determined by X-ray absorption spectroscopy. X-ray absorption spectroscopy (XAS) provides information about the local structure (approximately 5 A) of atoms surrounding the metal and has been widely used to characterize metalloproteins. The zinc site in native primase and in primase bound to low (30 mM) magnesium acetate was found to be tetrahedrally ligated by three sulfurs at an average distance of 2.36 +/- 0.02 A and one histidine nitrogen located at a distance of 2.15 +/- 0.03 A. When ATP, ATP and (dT)17, or ATP, low magnesium acetate and (dT)17 was added to primase, one (or two) additional nitrogen/oxygen ligands were coordinated to the zinc together with the histidine nitrogen at an average distance of 2.15 +/- 0.03 A. These additional ligands are likely from adjacent phosphates from ATP. Another structure was observed for the primase-(dT)17 complex in which an additional nitrogen/oxygen ligand likely from the phosphate backbone together with the histidine nitrogen was located at a significantly shorter average distance of 2.05 +/- 0.03 A. High magnesium acetate (300 mM) completely inactivates primase in a reversible manner such that the region near the zinc ligands becomes accessible to proteolytic digestion [Urlacher, T. M., and Griep, M. A. (1995) Biochemistry 34, 16708-16714]. In this inactive complex, additional oxygen/nitrogen ligands from acetate as well as the histidine nitrogen are located at a distance of 2.20 +/- 0.03 A from the zinc site. To test whether the catalytic magnesium was binding within approximately 5 A of the zinc, we incubated primase with high (300 mM) manganese acetate. The functional properties of magnesium and manganese are similar, but the larger atomic number of manganese enhances the X-ray backscattering, making it possible to identify. Since no significant difference was observed from the manganese-incubated sample, the catalytic metal-binding site is likely located >5 A from the zinc. These studies clearly show that primase zinc ligation changes upon binding substrates.