Molecular modeling of eluted peptides from DQ6 molecules (DQB1*0602 and DQB1*0604) negatively and positively associated with type 1 diabetes.

Insulin-dependent diabetes mellitus (IDDM) is positively associated with HLA-DQ8, DQ2, and DQ6 (B*0604) and negatively with DQ6 (B*0602). The mechanisms by which the DQ molecules control the development of IDDM is not known. DQ6 (B*0602) and DQ6 (B*0604) molecules share the same DQalpha chain but differ in the beta chain by six residues at positions 9, 30, 57, 70, 86, and 87. The aim of the study was to sequence the peptides eluted from both DQ6 molecules and to determine the binding motifs and construct peptides for docking them into the DQ6 peptide binding groove by molecular modeling. EBV transformed B cell line homozygous for DQ6 and hybridoma cell line secreting DQ6 specific antibody were grown in large-scale culture. The DQ6 molecules were precipitated with solid-phase bound antibodies specific for DQ6. The dissociation of peptides from MHC was done with ultrafiltration and separation of peptides by reversed-phase HPLC, using Edman degradation. A special application of Edman degradation is pool sequencing. This approach allowed us to determine common characteristics of all peptides associated with a given MHC molecule. The precipitation of DQ6 molecules and the peptide elution were done successfully. The sequencing of the peptides from DQ6 (B*0602) identified three fractions: (1) IINEPTAAAIAYGLD (Bovine HSP70), (2) IINEPTAAAIAGLDR (Human HSP70), and (3) NPRDAKACVVHGSDLK (Na+/K+ ATPase). Peptide eluted from DQ6 (B*0604) had a sequence ADLFRGTLDPVEK with sequence homology to HSP70 (residues 307-319). We were able to predict the motifs for DQ6 from the ligands eluted. We used molecular modeling as a tool to identify plausible binding motifs for peptides. Our studies show that peptide ADLFRGTLDPVEK and NPRDAKACVVHGSDLK fit well in the respective DQ6 binding grooves. These predicted motifs should then be useful for screening of autoantigens associated with diabetes and identifying the epitopes that are likely to interact with T cells.

Refinement of the structure of the ligand-occupied cholecystokinin receptor using a photolabile amino-terminal probe.

Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.

Ovalbumin(323-339) peptide binds to the major histocompatibility complex class II I-A(d) protein using two functionally distinct registers.

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway.

It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways. We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108. The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 A in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction. These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule. Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.

A peptide agonist acts by occupation of a monomeric G protein-coupled receptor: dual sites of covalent attachment to domains near TM1 and TM7 of the same molecule make biologically significant domain-swapped dimerization unlikely.

Membrane receptor dimerization is a well-established event for initiation of signaling at growth factor receptors and has been postulated to exist for G protein-coupled receptors, based on correction of nonfunctional truncated, mutant, or chimeric constructs by coexpression of appropriate normal complementary receptor domains. In this work, we have directly explored the molecular composition of the minimal functional unit of an agonist ligand and the wild-type G protein-coupled cholecystokinin (CCK) receptor, using photoaffinity labeling with a CCK analogue probe incorporating dual photolabile benzoylphenylalanine (Bpa) residues as sites of covalent attachment. This probe, 125I-D-Tyr-Gly-[(Nle28, 31, Bpa29,33)CCK-26-33], was shown to represent a full agonist and to specifically label the CCK receptor. Like probes incorporating individual photolabile residues in these positions,1,2 the two Bpa residues in the dual photoprobe covalently labeled receptor domains in the amino-terminal tail outside TM1 and in the third extracellular loop outside TM7. Absence of demonstrable receptor dimerization after the establishment of dual sites of covalent attachment supports the presence of these two domains within a single receptor molecule. Demonstration of the covalent adduct of a single probe molecule with the two cyanogen bromide fragments of the CCK receptor representing the expected domains further supports this interpretation. Thus, while domain-swapped dimerization of G protein-coupled receptors may be possible as a mechanism of rescue for nonfunctional molecules, it is not necessary for ligand binding and initiation of signaling at a wild-type receptor in this superfamily. The functional unit for CCK action is normally a ligand-receptor monomer.

Streptavidin-biotin binding energetics.

The high affinity energetics in the streptavidin-biotin system provide an excellent model system for studying how proteins balance enthalpic and entropic components to generate an impressive overall free energy for ligand binding. We review here concerted site-directed mutagenesis, biophysical, and computational studies of aromatic and hydrogen bonding interaction energetics between streptavidin and biotin. These results also have provided insight into how streptavidin builds a large activation barrier to dissociation by managing the enthalpic and entropic activation components. Finally, we review recent studies of the biotin dissociation pathway that address the fundamental question of how ligands exit protein binding pockets.

Structural basis of specificity and degeneracy of T cell recognition: pluriallelic restriction of T cell responses to a peptide antigen involves both specific and promiscuous interactions between the T cell receptor, peptide, and HLA-DR.

TCR engagement of peptide-MHC class II ligands involves specific contacts between the TCR and residues on both the MHC and peptide molecules. We have used molecular modeling and assays of peptide binding and T cell function to characterize these interactions for a CD4+ Th1 cell clone, ESL4.34, which recognizes a peptide epitope of the herpes simplex type 2 virus virion protein, VP16 393-405, in the context of several HLA-DR alleles. This clone responded to VP16 393-405 in proliferation and cytotoxicity assays when presented by DRB1*0402, DRB1*1102, and DRB1*1301, which share a common amino acid sequence, ILEDE, at residues 67-71 in the alpha-helical portion of the DRbeta polypeptide, but not when presented by other DR4, DR11, and DR13 alleles that are negative for this sequence. Using a panel of APCs expressing DR4 molecules that were mutagenized in vitro at individual residues within this shared epitope and using peptide analogues with single amino acid substitutions of predicted MHC and TCR contact residues, a unit of recognition was identified dependent on DRbeta residues 67-71 and relative position 4 (P4) of the VP16 393-405 peptide. The interactions of this portion of the peptide-DR ligand with the ESL4.34 TCR support a structural model for MHC-biased recognition in some Ag-specific and alloreactive T cell responses and suggest a possible mechanism for autoreactive T cell selection in rheumatoid arthritis.

A molecular model of myelin oligodendrocyte glycoprotein.

Myelin oligodendrocyte glycoprotein (MOG) is a protein on the surface of myelin sheaths. It is a putative target of the autoimmune attack in the inflammatory and demyelinating CNS disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. MOG belongs to the immunoglobulin superfamily (IgSF), and its extracellular N-terminal domain contains many conserved IgSF consensus residues seen in immunoglobulin variable region folds. The aim of the present study was to create a molecular model of the extracellular N-terminal domain of mouse MOG. No crystal structure is yet available of MOG, and thus a molecular model would be useful in providing insight into its structure and binding characteristics. Molecular graphics techniques and molecular dynamics with secondary structure-based restraints were used in the construction and refinement of the MOG model. Regions of high prediction confidence were identified, and possible glycosylation, dimerization, complement binding, and antibody-binding regions in MOG were mapped and analyzed.

Structural differences between HLA-DQ molecules associated with myasthenia gravis characterized by molecular modeling.

Myasthenia gravis (MG) is characterized by muscle weakness due to autoimmunity against the nicotinic acetylcholine receptor (nAChR). MG is associated with polymorphisms in HLA-DQ genes and the aim of the present study was to characterize structural differences in the peptide binding groove of HLA-DQ molecules positively and negatively associated with MG. Three dimensional models of the positively associated DQ2 (DQB1*02) and negatively associated DQ6 (DQB1*0603) molecules were constructed by homology modeling techniques. The differences in peptide binding properties were primarily localized to peptide-anchor pockets P7 and P9, which might be of importance for the binding of disease-associated peptides from the nAChR.

Alpha 1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists.

Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha1-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha 1A receptors were created by systematically substituting individual transmembrane domains from alpha 1D adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha 1A/D(TM2), and moderate reductions in affinity versus constructs alpha 1A/D(TM5), alpha 1A/B(TM5), and alpha 1A/D(TM6). Antagonist affinities for these chimeras exceeded those found for wild type alpha 1D and alpha 1B. Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha 1A receptor have a major impact on receptor subtype selectivity.

Direct identification of a second distinct site of contact between cholecystokinin and its receptor.

We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.

Direct identification of a distinct site of interaction between the carboxyl-terminal residue of cholecystokinin and the type A cholecystokinin receptor using photoaffinity labeling.

Mechanisms of ligand binding and activation of G protein-coupled receptors are particularly important, due to their ubiquitous expression and potential as drug targets. Molecular interactions between ligands and these receptors are best defined for small molecule ligands that bind within the transmembrane helices. Extracellular domains seem to be more important for peptide ligands, based largely on effects of receptor mutagenesis, where interference with binding or activity can reflect allosteric as well as direct effects. We now take the more direct approach of photoaffinity labeling the active site of the cholecystokinin (CCK) receptor, using a photolabile analogue of CCK having a blocked amino terminus. This probe, 125I-desaminotyrosyl-Gly-[Nle28,31, pNO2-Phe33]CCK-(26-33), binds specifically, saturably, and with high affinity (Ki = 3.3 nM) and has full agonist activity. This makes likely its being sited in a natural position within the receptor. As substrate, we used CHO-CCK receptor cells overexpressing functional recombinant rat type A CCK receptor. Covalent labeling of the appropriate Mr = 85,000-95,000 plasma membrane glycoprotein with core of Mr = 42,000 was established by SDS-polyacrylamide gel electrophoresis and autoradiography. A single domain adjacent to transmembrane 1 was labeled, as established by cyanogen bromide cleavage and separation by gel and/or high pressure liquid chromatography. The site of interaction was further defined by additional proteolysis with trypsin, with purification of the labeled fragment, followed by manual Edman degradation and radiochemical sequencing. This demonstrated that Trp39 was specifically labeled and likely resides proximate to the carboxyl-terminal pNO2-Phe33 residue of the probe. A model of this ligand-bound receptor has been constructed and will be used to plan future experiments to refine our understanding of this interaction.

Use of T cell receptor/HLA-DRB1*04 molecular modeling to predict site-specific interactions for the DR shared epitope associated with rheumatoid arthritis.

OBJECTIVE: To use molecular modeling tools to analyze the potential structural basis for the genetic association of rheumatoid arthritis (RA) with the major histocompatibility complex (MHC) "shared epitope," a set of conserved amino acid residues in the third hypervariable region of the DRbeta chain. METHODS: Homology model building techniques were used to construct molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor (TCR) from T cell clone EM025, which is specific for DR4 molecules containing the shared epitope sequence. Interactive graphics techniques were used to orient the TCR on the DR molecule, guided by surface complementarity analysis. RESULTS: The predicted TCR-MHC-peptide complex involved multiple interactions and specificity for the shared epitope. TCR residues CDR1beta D30, CDR2beta N51, and CDR3beta Q97 were positioned to potentially participate in hydrogen bond interactions with the shared epitope DRbeta residues Q70 and R71. CONCLUSION: These results suggest a structural mechanism in which specific TCR recognition and possibly Vbeta selection are directly influenced by the disease-associated MHC polymorphisms.

Analysis of critical residues of HLA-DQ6 molecules in insulin-dependent diabetes mellitus.

Among DQ6 molecules, DQA1*0102-DQB1*0602 is negatively associated with insulin-dependent diabetes mellitus (IDDM), but DQA1*0102-DQB1*0604 shows a neutral to positive association in Swedish children with IDDM. The aim of this study was to identify critical DQB1 residues that may account for the differences in IDDM association observed for these two DQ6 molecules. HLA-DQ genotyping in 425 IDDM patients and 367 matched controls showed DQ6 (B1*0602) in 1% of patients and 25% of controls (odds ratio (OR) 0.02). DQ6 (B1*0604) alone was neutral (9% of patients and 10% of controls) but in combination with DQ8, was positively associated (5% of patients, 1% of controls, OR 9.49). In both these DQ6 molecules the alpha-chain is the same but the beta-chain differs at positions 9, 30, 57, 70, 86 and 87. DQB1*0602 has F9, Y30, D57, G70, A86 and F87, whereas DQB1*0604 has Y9, H30, V57, R70, G86 and Y87. Three-dimensional models of the two DQ6 molecules, based on crystal coordinates of the homologous DR1 molecule, suggest that residue 57 beta will likely play a critical role in peptide-binding selectivity, whereas residue 70 beta is probably is major contact site for the T-cell receptor. The effects of these specific polymorphic substitutions in DQ molecules on peptide binding and T-cell receptor recognition may be significant in IDDM susceptibility.

Three-dimensional models for agonist and antagonist complexes with beta 2 adrenergic receptor.

Computer-modeling techniques have been used to generate docked complexes for a series of beta adrenergic agonists and antagonists with a three-dimensional model of the beta 2 adrenergic receptor. For all ligands tested, it proved possible to dock low-energy conformers in the receptor model, with sensible electrostatic, steric, and hydrogen-bonding interactions, many of which are supported by experimental studies of the beta 2 receptor. Our results illustrate the power of molecular modeling techniques, when coupled with appropriate experimental methods and data, to investigate structure-function properties of integral membrane receptor proteins that cannot yet be studied by direct structural methods.

Recognition of altered self major histocompatibility complex molecules modulated by specific peptide interactions.

Antigen-specific and major histocompatibility complex (MHC)-restricted recognition by the T cell receptor involves multiple structural contacts over a large molecular surface area. Using a human T cell clone specific for a rubella viral peptide restricted by subsets of HLA DR4 molecules, we identified structurally diverse combinations of peptide-MHC complexes which were functionally equivalent to T cell recognition. Presentation of the rubella-derived peptide on DR4 molecules with an E-74 polymorphism triggered T cell recognition, as did presentation of a single amino acid-substituted peptide in the context of DR4 molecule which lacked the E-74 site. Peptide binding and molecular modeling analysis indicates the structural and functional complementarity of T cell recognition for a specific amino acid side chain, whether contributed by the peptide or by the MHC molecule.

A structural model for TCR recognition of the HLA class II shared epitope sequence implicated in susceptibility to rheumatoid arthritis.

HLA molecules associated with rheumatoid arthritis (RA) contain a discrete structural element known as the shared epitope, a set of conserved amino acid residues located on the alpha helical portion of the class II beta chain. Each of the different HLA molecules associated with RA contain the same shared epitope sequence, although they may vary markedly in other regions of the class II structure, which also determine peptide-class II interactions. Previous mutagenesis studies and structural modelling indicate that key polymorphic amino acid side chains within the shared epitope sequence are in locations likely to contact the T cell receptor (TCR) during the trimolecular activation reaction between the HLA-peptide complex and TCR. We have evaluated the potential structural basis for such shared epitope recognition by analysing detailed molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor from T cell clone EM025, specific for HLA-DR4 molecules which carry the shared epitope. A likely orientation for the trimolecular complex was deduced in which the EM025 alpha chain interacts with the DR alpha chain and the EM025 beta chain interacts with the DR beta chain; residues Q70 and R71 within the DR beta chain shared epitope region are positioned for hydrogen bond interactions directly with Q97 of the TCR beta CDR3 region, D30 of the TCR beta CDR1 region, and possibly N51 of the TCR beta CDR2 region, indicating a degree of specific selection and interaction which encompasses multiple TCR contacts. These findings suggest a structural basis for the genetic associations with the HLA shared epitope and the potential contribution of this region to oligoclonal T cell selection and expansion in RA.

DR4 subtypes and their molecular properties in a population-based study of Swedish childhood diabetes.

The aim of this study was to determine the association between childhood insulin-dependent diabetes mellitus (IDDM) and HLA-DR4 subtypes and to test in a population-based investigation whether the DR4 association has an effect independent to that of DQ. First, HLA genotyping identified DR4 in 337/425 (79%) patients and 148/367 (40%) controls (Odds Ratio 5.67; p < 0.01). Second, a total of 14 DR4 subtypes were detected by PCR and sequence specific oligo probes. Only two DR4 subtypes, DRB1*0401 (62% patients and 25% controls; OR 4.95, p < 0.01) and *0404 (16% patients and 10% controls; OR 1.67, p < 0.05) were however positively associated with the disease. These two subtypes were positively associated only when linked to DQB1*0302-DQA1*0301 (DQ8) (56% patients and 14% controls; OR 7.69, p < 0.01; 15% patients and 10% controls; OR 1.55, p < 0.05, respectively). When DRB1*0401 was linked to DQB1*0301-DQA1*0301 (DQ7) (6% patients and 11% controls; OR 0.52, p < 0.05), this DR4 subtypes was negatively associated with IDDM. Third, tests of strongest association allowed the following ranking of alleles or haplotypes DQB1*0302-DQA1*0301 (DQ8) > DQB1*0302 > DRB1*0401 > DRB1*0404 and the association of DRB1*0401 has a significant effect in DQ8 positive IDDM patients. We conclude that the DR4 association with IDDM is secondary to DQ by linkage disequilibrium, which support the role of HLA-DQ as a primary genetic risk factor for IDDM.

Active site of bee venom phospholipase A2: the role of histidine-34, aspartate-64 and tyrosine-87.

In bee venom phospholipase A2, histidine-34 probably functions as a Brønsted base to deprotonate the attacking water. Aspartate-64 and tyrosine-87 form a hydrogen bonding network with histidine-34. We have prepared mutants at these positions and studied their kinetic properties. The mutant in which histidine-34 is changed to glutamine is catalytically inactive, while the mutants in which aspartate-64 is changed to asparagine or alanine (interfacial turnover numbers are reduced by 50-100-fold) or in which tyrosine-87 is changed to phenylalanine (no change in turnover number) retain good activity. The interfacial Michaelis constants are changed by less than 10-fold for all mutants. Molecular simulations suggest that mutation of aspartate-64 and tyrosine-87 should yield enzymes that retain a native-like structure and support catalysis. The pKa of the histidine-34 imidazole was deduced from the pH-rate profile and from the pH dependence of the rate of histidine-34 alkylation by 2-bromo-4'-nitroacetophenone. The pKa is increased about one-half unit by the tyrosine-87 mutation and reduced about one-half unit by the aspartate-64 to asparagine mutation, while in the aspartate-64 to alanine mutant the pKa is unchanged. These pKas are generally consistent with results of electrostatic calculations and suggest that the hydrogen bond between aspartate-64 and histidine-34 is not unusually strong. The hydrogen bonding network linking tyrosine-87 to aspartate-64 and aspartate-64 to histidine-34 is not critical for catalysis.

MD Display: an interactive graphics program for visualization of molecular dynamics trajectories.

MD Display was developed as a means of visualizing molecular dynamic trajectories generated by Amber. The program runs on Silicon Graphics workstations, and features a simple user interface, and convenient display and analysis options. The program has now been extended to accept input from several other molecular dynamics programs.