Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.     

Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. Implications for the catalytic mechanisms

Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Tyrosine 387 and arginine 404 are critical in the hydrolytic mechanism of Escherichia coli aminopeptidase P

Analysis of the pH-rate profile for catalysis of bradykinin cleavage by aminopeptidase P (AMPP), a manganese-containing hydrolase from Escherichia coli, was carried out to show that optimal catalytic function is obtained at neutral pH. On the basis of information derived from the crystal structure, peptidase sequence alignments, and the hydrolysis of organophosphate triesters, active site residues Arg153, Arg370, Trp88, Tyr387, and Arg404 were identified as potential catalytic residues. Site-directed mutagenesis was used to substitute these residues with Leu, Ala, Trp, Lys, or Phe. The kcat values for the Arg153, Arg370, and Trp88 mutants were nearly the same as that for the wild-type enzyme. The kcat values of the R404K, R404A, and Y387A mutants were lower by factors of 285, 400, and 16, respectively. Inductively coupled plasma mass spectrometry and circular dichroism spectroscopy showed that Arg404 is not required for metal chelation or stabilization of protein secondary structure. The hydrogen bond network observed between the side chains of conserved residues Asp260, Arg404, and Tyr387 indicated that Arg404 participates in proton relay. This was further evidenced by the return of activity in the R404A mutant by the addition of guanidine. Also, reduced catalytic efficiency in the R404K mutant, which conserves the positive charge at the bridge site, shows that only the arginine group of Arg404 (not the ammonium group of Lys404) can participate in the hydrogen bond network. The hydrogen bond interaction between the Arg404 and the Tyr387 ring hydroxyl group is suggested by the reduced catalytic efficiency of the Y387F mutant.     

Structural and kinetic contributions of the oxyanion binding site to the catalytic activity of acylaminoacyl peptidase

It is widely accepted that the catalytic activity of serine proteases depends primarily on the Asp-His-Ser catalytic triad and other residues within the vicinity of this motif. Some of these residues form the oxyanion binding site that stabilizes the tetrahedral intermediate by hydrogen bonding to the negatively charged oxyanion. In acylaminoacyl peptidase from the thermophile Aeropyrum pernix, the main chain NH group of Gly369 is one of the hydrogen bond donors forming the oxyanion binding site. The side chain of His367, a conserved residue in acylaminoacyl peptidases across all species, fastens the loop holding Gly369. Determination of the crystal structure of the H367A mutant revealed that this loop, including Gly369, moves away considerably, accounting for the observed three orders of magnitude decrease in the specificity rate constant. For the wild-type enzyme ln(k(cat)/K(m)) vs. 1/T deviates from linearity indicating greater rate enhancement with increasing temperature for the dissociation of the enzyme-substrate complex compared with its decomposition to product. In contrast, the H367A variant provided a linear Arrhenius plot, and its reaction was associated with unfavourable entropy of activation. These results show that a residue relatively distant from the active site can significantly affect the catalytic activity of acylaminoacyl peptidase without changing the overall structure of the enzyme.     

Shifting the optimum pH of Bacillus circulans xylanase towards acidic side by introducing arginine

Electrostatic interactions are important in protein folding, binding, flexibility, stability and function. The pH at which the enzyme is maximally active is determined by the pK_as of the active site residues, which are modulated by several factors including the change in electrostatics in its vicinity. As the acidic xylanases are important in food and animal feed industries, electrostatic interactions are introduced in Bacillus circulans xylanase to shift their pH optima towards the acidic side. Arg substitutions are made to modulate the pKas of the active site residues. Neutral residues are substituted by Arg in such a way that the substituted residue can make direct interaction with the catalytic residues. However, the mutations with other titratable residues (Asp, Arg, Lys, His, Tyr, and Ser) present in between the catalytic sites and the substituted sites are avoided. Site directed mutagenesis was conducted to confirm the strategy. The results show the shift in pH optima of the mutants towards the acidic side by 0.5–1.5 unit. Molecular dynamics simulation of the mutant V37R reveals that the decrease in activity is due to the increase in distance between the substrate oxygen atoms and catalytic glutamates.     

Mutational analysis of the tetrahydrobiopterin-binding site in inducible nitric-oxide synthase.

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.     

Mutation of residues critical for benzohydroxamic acid binding to horseradish peroxidase isoenzyme C.

Aromatic substrate binding to peroxidases is mediated through hydrophobic and hydrogen bonding interactions between residues on the distal side of the heme and the substrate molecule. The effects of perturbing these interactions are investigated by an electronic absorption and resonance Raman study of benzohydroxamic acid (BHA) binding to a series of mutants of horseradish peroxidase isoenzyme C (HRPC). In particular, the Phe179 --> Ala, His42 --> Glu variants and the double mutant His42 --> Glu:Arg38 --> Leu are studied in their ferric state at pH 7 with and without BHA. A comparison of the data with those previously reported for wild-type HRPC and other distal site mutants reaffirms that in the resting state mutation of His42 leads to an increase of 6-coordinate aquo heme forms at the expense of the 5-coordinate heme state, which is the dominant species in wild-type HRPC. The His42Glu:Arg38Leu double mutant displays an enhanced proportion of the pentacoordinate heme state, similar to the single Arg38Leu mutant. The heme spin states are insensitive to mutation of the Phe179 residue. The BHA complexes of all mutants are found to have a greater amount of unbound form compared to the wild-type HRPC complex. It is apparent from the spectral changes induced on complexation with BHA that, although Phe179 provides an important hydrophobic interaction with BHA, the hydrogen bonds formed between His42 and, in particular, Arg38 and BHA assume a more critical role in the binding of BHA to the resting state.     

A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase

The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.     

Molecular Dynamics Assisted Mechanistic Study of Isoniazid-Resistance against Mycobacterium tuberculosis InhA

Examination of InhA mutants I16T, I21V, I47T, S94A, and I95P showed that direct and water mediated H-bond interactions between NADH and binding site residues reduced drastically. It allowed conformational flexibility to NADH, particularly at the pyrophosphate region, leading to weakening of its binding at dinucleotide binding site. The highly scattered distribution of pyrophosphate dihedral angles and chi1 side chain dihedral angles of corresponding active site residues therein confirmed weak bonding between InhA and NADH. The average direct and water mediated bridged H-bond interactions between NADH and mutants were observed weaker as compared to wild type. Further, estimated NADH binding free energy in mutants supported the observed weakening of InhA-NADH interactions. Similarly, per residue contribution to NADH binding was also found little less as compared to corresponding residues in wild type. This investigation clearly depicted and supported the effect of mutations on NADH binding and can be accounted for isoniazid resistance as suggested by previous biochemical and mutagenic studies. Further, structural analysis of InhA provided the crucial points to enhance the NADH binding affinity towards InhA mutants in the presence of direct InhA inhibitors to combat isoniazid drug resistance. This combination could be a potential alternative for treatment of drug resistant tuberculosis.     

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.     

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only α, α′- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has D312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (D322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity.     

An arginine residue instead of a conserved leucine residue in the recognition helix of the finger 3 of Zif268 stabilizes the domain structure and mediates DNA binding

The Cys(2)His(2)-type zinc finger is a common DNA binding motif that is widely used in the design of artificial zinc finger proteins. In almost all Cys(2)His(2)-type zinc fingers, position 4 of the α-helical DNA-recognition site is occupied by a Leu residue involved in formation of the minimal hydrophobic core. However, the third zinc finger domain of native Zif268 contains an Arg residue instead of the conserved Leu. Our aim in the present study was to clarify the role of this Arg in the formation of a stable domain structure and in DNA binding by substituting it with a Lys, Leu, or Hgn, which have different terminal side-chain structures. Assessed were the metal binding properties, peptide conformations, and DNA-binding abilities of the mutants. All three mutant finger 3 peptides exhibited conformations and thermal stabilities similar to the wild-type peptide. In DNA-binding assays, the Lys mutant bound to target DNA, though its affinity was lower than that of the wild-type peptide. On the other hand, the Leu and Hgn mutants had no ability to bind DNA, despite the similarity in their secondary structures to the wild-type. Our results demonstrate that, as with the Leu residue, the aliphatic carbon side chain of this Arg residue plays a key role in the formation of a stable zinc finger domain, and its terminal guanidinium group appears to be essential for DNA binding mediated through both electrostatic interaction and hydrogen bonding with DNA phosphate backbone.     

Structure of the hirugen and hirulog 1 complexes of alpha-thrombin

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.     

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Highly organized but pliant active site of DNA polymerase beta: compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses

To link conformational transitions noted for DNA polymerases with kinetic results describing catalytic efficiency and fidelity, we investigate the role of key DNA polymerase beta residues on subdomain motion through simulations of five single-residue mutants: Arg-283-Ala, Tyr-271-Ala, Asp-276-Val, Arg-258-Lys, and Arg-258-Ala. Since a movement toward a closed state was only observed for R258A, we suggest that Arg(258) is crucial in modulating motion preceding chemistry. Analyses of protein/DNA interactions in the mutant active site indicate distinctive hydrogen bonding and van der Waals patterns arising from compensatory structural adjustments. By comparing closed mutant complexes with the wild-type enzyme, we interpret experimentally derived nucleotide binding affinities in molecular terms: R283A (decreased), Y271A (increased), D276V (increased), and R258A (decreased). Thus, compensatory interactions (e.g., in Y271A with adjacent residues Phe(272), Asn(279), and Arg(283)) increase the overall binding affinity for the incoming nucleotide although direct interactions may decrease. Together with energetic analyses, we predict that R258G might increase the rate of nucleotide insertion and maintain enzyme fidelity as R258A; D276L might increase the nucleotide binding affinity more than D276V; and R283A/K280A might decrease the nucleotide binding affinity and increase misinsertion more than R283A. The combined observations regarding key roles of specific residues (e.g., Arg(258)) and compensatory interactions echo the dual nature of polymerase active site, namely versatility (to accommodate various basepairs) and specificity (for preserving fidelity) and underscore an organized but pliant active site essential to enzyme function.     

Energetics of hydrogen bond switch, residue burial and cavity analysis reveals molecular basis of improved heparin binding to antithrombin

Antithrombin III (ATIII) is the main inhibitor of the coagulation proteases like factor Xa and thrombin. Anticoagulant activity of ATIII is increased by several thousand folds when activated by vascular wall heparan sulfate proteoglycans (HSPGs) and pharmaceutical heparins. ATIII isoforms in human plasma, alpha-ATIII and beta-ATIII differ in the amount of glycosylation which is the basis of differences in their heparin binding affinity and function. Crystal structures and site directed mutagenesis studies have mapped the heparin binding site in ATIII, however the hydrogen bond switch and energetics of interaction during the course of heparin dependent conformational change remains largely unclear. An analysis of heparin bound conformational states of ATIII using PEARLS software showed that in heparin bound intermediate state, Arg 47 and Arg 13 residues make hydrogen bonds with heparin but in the activated conformation Lys 11 and Lys 114 have more hydrogen bond interactions. In the protease bound-antithrombin-pentasaccharide complex Lys 114, Pro 12 and Lys 125 form important hydrogen bonding interactions. The results showed that A-helix and N-terminal end residues are more important in the initial interactions but D-helix is more important during the latter stage of conformational activation and during the process of protease inhibition. We carried out the residue wise Accessible Surface Area (ASA) analysis of alpha and beta ATIII native states and the results indicated major differences in burial of residues from Ser 112 to Ser 116 towards the N-terminal end. This region is involved in the P-helix formation on account of heparin binding. A cavity analysis showed a progressively larger cavity formation during activation in the region just adjacent to the heparin binding site towards the C-terminal end. We hypothesize that during the process of conformational change after heparin binding beta form of antithrombin has low energy barrier to form D-helix extension toward N and C-terminal end as compared to alpha isoform.     

Role of hydrogen bonding in the interaction between a xylan binding module and xylan

NMR studies of the internal family 2b carbohydrate binding module (CBM2b-1) of Cellulomonas fimi xylanase 11A have identified six polar residues and two aromatic residues that interact with its target ligand, xylan. To investigate the importance of the various interactions, free energy and enthalpy changes have been measured for the binding of xylan to native and mutant forms of CBM2b-1. The data show that the two aromatic residues, Trp 259 and Trp 291, play a critical role in the binding, and similarly that mutants N264A and T316A have no affinity for the xylose polymer. Interestingly, mutations E257A, Q288A, N292A, E257A/Q288A, E257A/N292A, and E257A/N292A/Q288A do not significantly diminish the affinity of CBM2b-1 for the xylose polymers, but do influence the thermodynamics driving the protein-carbohydrate interactions. These thermodynamic parameters have been interpreted in light of a fresh understanding of enthalpy-entropy compensation and show the following. (1) For proteins whose ligands are bound on an exposed surface, hydrogen bonding confers little specificity or affinity. It also displays little cooperativity. Most specificity and affinity derive from binding between the face of sugar rings and aromatic rings. (2) Loss of hydrogen bonding interactions leads to a redistribution of the remaining bonding interactions such that the entropic mobility of the ligand is maximized, at the expense (if necessary) of enthalpically favorable bonds. (3) Changes in entropy and enthalpy in the binding between polysaccharide and a range of mutants can be interpreted by considering changes in binding and flexibility, without any need to consider solvent reorganization.     

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein

ATP:Cobalamin adenosyltransferases catalyze the transfer a 5′-deoxyadenosyl moiety from ATP to cob(I)alamin in the synthesis of the Co–C bond of coenzyme B₁₂. There are three types of adenosyltransferases, CobA, PduO and EutT. Among these adenosyltransferases, the PduO-type adenosyltransferases is the most widely distributed enzyme. Structural comparisons between apo BcPduO and BcPduO in complex with MgATP revealed that the N-terminal strands of both structures were ordered, which is in contrast with the most previously available PduO-type adenosyltransferase structures. Furthermore, unlike other reported structures, apo BcPduO was bound to additional dioxane molecules causing a side chain conformational change at the Tyr30 residue, which is an important residue that mediates hydrogen bonding with ATP molecules upon binding of cobalamin to the active site. This study provides more structural information into the role of active site residues on substrate binding.     

Geometry of nonbonded interactions involving planar groups in proteins

Although hydrophobic interaction is the main contributing factor to the stability of the protein fold, the specificity of the folding process depends on many directional interactions. An analysis has been carried out on the geometry of interaction between planar moieties of ten side chains (Phe, Tyr, Trp, His, Arg, Pro, Asp, Glu, Asn and Gln), the aromatic residues and the sulfide planes (of Met and cystine), and the aromatic residues and the peptide planes within the protein tertiary structures available in the Protein Data Bank. The occurrence of hydrogen bonds and other nonconventional interactions such as C–H⋯π, C–H⋯O, electrophile–nucleophile interactions involving the planar moieties has been elucidated. The specific nature of the interactions constraints many of the residue pairs to occur with a fixed sequence difference, maintaining a sequential order, when located in secondary structural elements, such as α-helices and β-turns. The importance of many of these interactions (for example, aromatic residues interacting with Pro or cystine sulfur atom) is revealed by the higher degree of conservation observed for them in protein structures and binding regions. The planar residues are well represented in the active sites, and the geometry of their interactions does not deviate from the general distribution. The geometrical relationship between interacting residues provides valuable insights into the process of protein folding and would be useful for the design of protein molecules and modulation of their binding properties.