Arg Side Chain Research Articles

Alpha-helical stabilization by side chain shielding of backbone hydrogen bonds.

We study atomic models of the thermodynamics of the structural transition of peptides that form alpha-helices. The effect of sequence variation on alpha-helix formation for alanine-rich peptides, Ac-Ala21-methyl amide (A21) and Ac-A5 (AAARA)3A-methyl amide (Fs peptide), is investigated by atomic simulation studies of the thermodynamics of the helix-coil transition in explicit water. The simulations show that the guanidinium group in the Arg side chains in the Fs peptide interacts with the carbonyl group four amino acids upstream in the chain and desolvates backbone hydrogen bonds. This desolvation can be directly correlated with a higher probability of hydrogen bond formation. We find that Fs has higher helical content than A21 at all temperatures. A small modification in the amber force field reproduces the experimental helical content and helix-coil transition temperatures for the Fs peptide.

Probing ligand recognition in the decarboxylase antibody 21D8: implications for the catalytic mechanism.

Antibody 21D8, which was elicited with a naphthalene-1,5-disulfonate hapten, catalyzes the medium-sensitive decarboxylation of 5-nitro-3-carboxybenzisoxazole. Structural studies on the hapten-antibody complex show that the active site contains two anion binding pockets separated by a hydrophobic region. To gain further insight into the ligand binding and catalytic mechanism of 21D8, six site-directed mutants were prepared, four for investigating the role of each of the two hapten sulfonate binding sites and two for examining packing interactions between bound ligands and the binding pocket. With the exception of an Arg(L46)Met substitution in the more deeply buried sulfonate binding pocket, modification of the active site resulted in reductions in catalytic efficiency (k(cat)/k(uncat)), ranging between 3- and 23-fold. Importantly, and contrary to predictions based on computational docking experiments, the differential effects of the individual mutations on the K(m), K(TS), and K(product) parameters suggest that only substrate binding modes which place the carboxylate group in the more solvent-exposed sulfonate binding site are catalytically relevant. Such an orientation would permit a potentially significant interaction between the developing oxyanion in the transition state and the side chain of Arg(L96). Incomplete desolvation of the carboxylate in this orientation may also help explain the modest efficiency of 21D8 compared to the most accelerating aprotic dipolar organic solvents.

Crystal structure of an antigen-binding fragment bound to single-stranded DNA

Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 Å crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops.

Folded Monomer of HIV-1 Protease

The mature human immunodeficiency virus type 1 protease rapidly folds into an enzymatically active stable dimer, exhibiting an intricate interplay between structure formation and dimerization. We now show by NMR and sedimentation equilibrium studies that a mutant protease containing the R87K substitution (PR(R87K)) within the highly conserved Gly(86)-Arg(87)-Asn(88) sequence forms a monomer with a fold similar to a single subunit of the dimer. However, binding of the inhibitor DMP323 to PR(R87K) produces a stable dimer complex. Based on the crystal structure and our NMR results, we postulate that loss of specific interactions involving the side chain of Arg(87) destabilizes PR(R87K) by perturbing the inner C-terminal beta-sheet (residues 96-99 from each monomer), a region that is sandwiched between the two beta-strands formed by the N-terminal residues (residues 1-4) in the mature protease. We systematically examined the folding, dimerization, and catalytic activities of mutant proteases comprising deletions of either one of the terminal regions (residues 1-4 or 96-99) or both. Although both N- and C-terminal beta-strands were found to contribute to dimer stability, our results indicate that the inner C-terminal strands are absolutely essential for dimer formation. Knowledge of the monomer fold and regions critical for dimerization may aid in the rational design of novel inhibitors of the protease to overcome the problem of drug resistance.

Interaction of acetylene and cyanide with the resting state of nitrogenase alpha-96-substituted MoFe proteins.

The nitrogenase MoFe protein contains the active site metallocluster called FeMo-cofactor [7Fe-9S-Mo-homocitrate] that exhibits an S = 3/2 EPR signal in the resting state. No interaction with FeMo-cofactor is detected when either substrates or inhibitors are incubated with MoFe protein in the resting state. Rather, the detection of such interactions requires the incubation of the MoFe protein together with its obligate electron donor, called the Fe protein, and MgATP under turnover conditions. This indicates that a more reduced state of the MoFe protein is required to accommodate substrate or inhibitor interaction. In the present work, substitution of an arginine residue (alpha-96(Arg)) located next to the active site FeMo-cofactor in the MoFe protein by leucine, glutamine, alanine, or histidine is found to result in MoFe proteins that can interact with acetylene or cyanide in the as-isolated, resting state without the need for the Fe protein, or MgATP. The dithionite-reduced, resting states of the alpha-96(Leu)-, alpha-96(Gln)-, alpha-96(Ala)-, or alpha-96(His)-substituted MoFe proteins show an S = 3/2 EPR signal (g = 4.26, 3.67, 2.00) similar to that assigned to FeMo-cofactor in the wild-type MoFe protein. However, in contrast to the wild-type MoFe protein, the alpha-96-substituted MoFe proteins all exhibit changes in their EPR spectra upon incubation with acetylene or cyanide. The alpha-96(Leu)-substituted MoFe protein was representative of the other alpha-96-substituted MoFe proteins examined. The incubation of acetylene with the alpha-96(Leu) MoFe protein decreased the intensity of the normal FeMo-cofactor signal with the appearance of a new EPR signal having inflections at g = 4.50 and 3.50. Incubation of cyanide with the alpha-96(Leu) MoFe protein also decreased the FeMo-cofactor EPR signal with concomitant appearance of a new EPR signal having an inflection at g = 4.06. The acetylene- and cyanide-dependent EPR signals observed for the alpha-96(Leu)-substituted MoFe protein were found to follow Curie law 1/T dependence, consistent with a ground-state transition as observed for FeMo-cofactor. The microwave power dependence of the EPR signal intensity is shifted to higher power for the acetylene- and cyanide-dependent signals, consistent with a change in the relaxation properties of the spin system of FeMo-cofactor. Finally, the alpha-96(Leu)-substituted MoFe protein incubated with (13)C-labeled cyanide displays a (13)C ENDOR signal with an isotropic hyperfine coupling of 0.42 MHz in Q-band Mims pulsed ENDOR spectra. This indicates the existence of some spin density on the cyanide, and thus suggests that the new component of the cyanide-dependent EPR signals arise from the direct bonding of cyanide to the FeMo-cofactor. These data indicate that both acetylene and cyanide are able to interact with FeMo-cofactor contained within the alpha-96-substituted MoFe proteins in the resting state. These results support a model where effective interaction of substrates or inhibitors with FeMo-cofactor occurs as a consequence of both increased reactivity and accessibility of FeMo-cofactor under turnover conditions. We suggest that, for the wild-type MoFe protein, the alpha-96(Arg) side chain acts as a gatekeeper, moving during turnover in order to permit accessibility of acetylene or cyanide to a specific [4Fe-4S] face of FeMo-cofactor.

Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin.

The side chain of residue Arg(238) in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg(238) in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg(238)-->Lys), the positive charge was removed (Arg(238)-->Met) or the charge was reversed (Arg(238)-->Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E(12)) for each enzyme fall within the region of -247+/-10 mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme-NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy-enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

A G577R Mutation in the Human AR P Box Results in Selective Decreases in DNA Binding and in Partial Androgen Insensitivity Syndrome

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the Cβ of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.

The crystal structure of chorismate lyase shows a new fold and a tightly retained product.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

Different susceptibility to oxidation of proline and arginine residues of apolipoprotein B-100 among subspecies of low density lipoproteins

γ-Glutamyl semialdehyde is a primary oxidation product of apolipoprotein (apo) B-100 proline (Pro) and arginine (Arg) side chain residues. By reduction γ-glutamyl semialdehyde forms 5-hydroxy-2-aminovaleric acid (HAVA). Here we describe the application of sensitive and specific HAVA measurement to characterize the formation of γ-glutamyl semialdehyde in several domains of apoB-100 in LDL 1 (S f 7–12) and LDL 2 (S f 0–7) subfractions subjected to oxidative damage in the presence of iron in vitro. Results suggest that susceptibility of apoB-100 Pro and Arg residues toward oxygen radicals drastically changes along the lipoprotein metabolic cascade.

Peptide-triggered conformational switch in HIV-1 RRE RNA complexes.

We have used NMR spectroscopy to determine the solution structure of a complex between an oligonucleotide derived from stem IIB of the Rev responsive element (RRE-IIB) of HIV-1 mRNA and an in vivo selected, high affinity binding Arg-rich peptide. The peptide binds in a partially alpha-helical conformation into a pocket within the RNA deep groove. Comparison with the structure of a complex between an alpha-helical Rev peptide and RRE-IIB reveals that the sequence of the bound peptide determines the local conformation of the RRE peptide binding site. A conformational switch of an unpaired uridine base was revealed; this points out into the solvent in the Rev peptide complex, but it is stabilized inside the RNA deep groove by stacking with an Arg side chain in the selected peptide complex. The conformational switch has been visualized by NMR chemical shift mapping of the uridine H5/H6 atoms during a competition experiment in which Rev peptide was displaced from RRE-IIB by the higher affinity binding selected peptide.

Amino acid type-selective backbone 1H-15N-correlations for Arg and Lys.

Four novel amino acid type-selective triple resonance experiments to identify the backbone amino proton and nitrogen resonances of Arg and Lys and of their sequential neighbors in (13C,15N)-labeled proteins are presented: the R(i+1)-HSQC and R(i,i+1)-HSQC select signals originating from Arg side chains, the K(i+1)-HSQC and K(i,i+1)-HSQC select signals originating from Lys side chains. The selection is based on exploiting the characteristic chemical shifts of a pair of carbon atoms in Arg and Lys side chains using selective 90 degree pulses. The new experiments are recorded as two-dimensional 1H-15N-correlations and their performance is demonstrated with the application to a protein domain of 83 amino acids.

Structure and Site-directed Mutagenesis of a Flavoprotein fromEscherichia coli That Reduces Nitrocompounds: ALTERATION OF PYRIDINE NUCLEOTIDE BINDING BY A SINGLE AMINO ACID SUBSTITUTION

The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.

Fusarium oxysporumtrypsin at atomic resolution at 100 and 283 K: a study of ligand binding

The X-ray structure of F. oxysporum trypsin has been determined at atomic resolution, revealing electron density in the binding site which was interpreted as a peptide bound in the sites S1, S2 and S3. The structure, which was initially determined at 1.07 A resolution and 283 K, has an Arg in the S1 specificity pocket. The study was extended to 0.81 A resolution at 100 K using crystals soaked in Arg, Lys and Gln to study in greater detail the binding at the S1 site. The electron density in the binding site was compared between the different structures and analysed in terms of partially occupied and overlapping components of peptide, solvent water and possibly other chemical moieties. Arg-soaked crystals reveal a density more detailed but similar to the original structure, with the Arg side chain visible in the S1 pocket and residual peptide density in the S2 and S3 sites. The density in the active site is complex and not fully interpreted. Lys at high concentrations displaces Arg in the S1 pocket, while some main-chain density remains in sites S2 and S3. Gln has been shown not to bind. The free peptide in the S1-S3 sites binds in a similar way to the binding loop of BPTI or the inhibitory domain of the Alzheimer's beta-protein precursor, with some differences in the S1 site.

Design, synthesis, and modeling of novel cyclic thrombin receptor-derived peptide analogues of the Ser42-Phe-Leu-Leu-Arg46 motif sequence with fixed conformations of pharmacophoric groups: importance of a Phe/Arg/NH2 cluster for receptor activation and implications in the design of nonpeptide thrombin receptor mimetics.

The novel cyclic analogues cyclo(Phe-Leu-Leu-Arg-epsilonLys-Dap) (1) and cyclo(D-Phe-Leu-Leu-Arg-epsilonLys-Dap) (2), which differ only in the absolute conformation of Phe, have been designed and synthesized based upon the minimal peptide sequence Phe-Leu-Leu-Arg which has been found to exhibit biological activity for the thrombin receptor. Compound 1, in which all amino acids have the L-configuration, exhibited higher activity in the rat aorta relaxation and rat longitudinal muscle bioassays compared to compound 2, in which the Phe residue is in the D-configuration. This is attributed to the spatial proximity of the Phe and Arg in compound 1 which does not exist in its diastereomeric compound 2, as is depicted from a combination of NMR studies and computational analysis. Structure-activity studies (SAR) showed that the Phe and Arg side chains along with a primary amino group form an active recognition motif that is augmented by the presence of a second primary amino group in the cyclic peptide. We suggest that a comparable cyclic conformation may be responsible for the interaction of linear TRAPs with the thrombin receptor. The validity of this proposition was tested by the synthesis of four active nonpeptide thrombin receptor mimetics. Substance (S)-N-(6-guanidohexanoyl)-N'-(2-amino-3-phenylpropionyl)piperazine (3), in which the pharmacophoric phenyl, guanidino, and amino groups were incorporated onto a piperazine template, was found to be the most active compared to the other synthesized compounds which lack the amino pharmacophoric group.

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

Structural studies of lysyl-tRNA synthetase: conformational changes induced by substrate binding.

Lysyl-tRNA synthetase is a member of the class II aminoacyl-tRNA synthetases and catalyses the specific aminoacylation of tRNA(Lys). The crystal structure of the constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli has been determined to 2.7 A resolution in the unliganded form and in a complex with the lysine substrate. A comparison between the unliganded and lysine-bound structures reveals major conformational changes upon lysine binding. The lysine substrate is involved in a network of hydrogen bonds. Two of these interactions, one between the alpha-amino group and the carbonyl oxygen of Gly 216 and the other between the carboxylate group and the side chain of Arg 262, trigger a subtle and complicated reorganization of the active site, involving the ordering of two loops (residues 215-217 and 444-455), a change in conformation of residues 393-409, and a rotation of a 4-helix bundle domain (located between motif 2 and 3) by 10 degrees. The result of these changes is a closing up of the active site upon lysine binding.

Probing the Catalytic Mechanism of the Insulin Receptor Kinase with a Tetrafluorotyrosine-containing Peptide Substrate

The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK(a) of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzyme-substrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp(1132) and Arg(1136), consistent with elevation of the pK(a). These findings strongly support a reaction mechanism favoring a dissociative transition state.

The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity.

Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes. It is a processing enzyme, requiring extended peptide substrates containing an Asp residue. The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor. The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B. An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile. The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.

Thermodynamic Stability of HLA-B*2705·Peptide Complexes: EFFECT OF PEPTIDE AND MAJOR HISTOCOMPATIBILITY COMPLEX PROTEIN MUTATIONS

Thermodynamic Stability of HLA-B*2705·Peptide Complexes: EFFECT OF PEPTIDE AND MAJOR HISTOCOMPATIBILITY COMPLEX PROTEIN MUTATIONS

Theoretical Calculation of pKaReveals an Important Role of Arg205 in the Activity and Stability of Streptomyces sp. N174 Chitosanase

Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.