Saturation mutagenesis reveals that GLU54 of norovirus 3C-like protease is not essential for the proteolytic activity

The norovirus 3C-like protease is a member of the chymotrypsin-like serine protease superfamily. Previous characterization of its crystal structure has implicated the Glu54-His30-Cys139 triad in the catalysis. In the present study, the Glu54 residue of the protease was subjected to site-saturation mutagenesis, with the result that nearly half of the mutants retained the significant proteolytic activity. It was suggested that a carboxylate at position 54 was not essential for the activity. The in vitro assays of the proteolysis revealed that most of Glu54 mutants retained relatively high proteolytic activity. When the Glu54 mutation was combined with the Ser mutation of the Cys139 residue, a nucleophile, only the Asp54 and Gln54 mutations showed proteolytic activity comparable to that of the Ser139 single mutant, suggesting that a hydrogen bond between Glu54 and His30 was critical in the Ser139 background. These results suggested that the mechanism of the proteolysis by the wild-type norovirus 3C-like protease was different from that of typical chymotrypsin-like serine proteases.     

Human rhinovirus 3C protease: a cysteine protease showing the trypsin(ogen)-like fold

Viral-encoded proteases cleave precursor polyprotein(s) leading to maturation of infectious virions. Strikingly, human rhinovirus 3C protease shows the trypsin(ogen)-like serine protease fold based on two topologically equivalent six-stranded β-barrels, but displays residue Cys147 as the active site nucleophile. By contrast, papain, which is representative of most cysteine proteases, does not display the trypsin(ogen)-like fold. Remarkably, in human rhinovirus 3C cysteine protease, the catalytic residues Cys147, His40 and Glu71 are positioned as Ser195, His57 and Asp102, respectively, building up the catalytic triad of serine proteases in the chymotrypsin–trypsin–elastase family. However, as compared to trypsin-like serine proteases and their zymogens, residue His40 and the oxyanion hole of human rhinovirus 3C cysteine protease, both key structural components of the active site, are located closer to the protein core. Human rhinovirus 3C cysteine protease cleaves preferentially GlnGly peptide bonds or, less commonly, the GlnSer, GlnAla, GluSer or GluGly pairs. Finally, human rhinovirus 3C cysteine protease and the 3CD cysteine protease–polymerase covalent complex bind the 5′ non-coding region of rhinovirus genomic RNA, an essential function for replication of the viral genome.     

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.     

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.     

Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site

Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.     

Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin

The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).     

Proteolysis of an active site peptide of lactate dehydrogenase by human immunodeficiency virus type 1 protease.

The muscle and heart lactate dehydrogenase (LDHs) of rabbit and pig are specifically cleaved at a single position by HIV-1 protease, resulting in the conversion of 36-kDa subunits of the oligomeric enzymes into 21- and 15-kDa protein bands as analyzed by SDS-PAGE. While the proteolysis was observed at neutral pH, it became more pronounced at pH 6.0 and 5.0. The time courses of the cleavage of the 36-kDa subunits were commensurate with the time-dependent loss of both quaternary structure and enzymatic activity. These results demonstrated that deoligomerization of rabbit muscle LDH at acidic pH rendered its subunits more susceptible to proteolysis, suggesting that a partially denatured form of the enzyme was the actual substrate. Proteolytic cleavage of the rabbit muscle enzyme occurred at a decapeptide sequence, His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp (scissile bond denoted throughout by an asterisk), which constitutes a "strand-loop" element in the muscle and heart LDH structures and contains the active site histidyl residue His-193. The kinetic parameters Km, Vmax/KmEt, and Vmax/Et for rabbit muscle LDH and the synthetic decapeptide Ac-His-Gly-Trp-Ile-Leu*Gly-Glu-His-Gly-Asp-NH2 were nearly identical, suggesting that the decapeptide within the protein substrate is conformationally mobile, as would be expected for the peptide substrate in solution. Insertion of part of this decapeptide sequence into bacterial galactokinase likewise rendered this protein susceptible to proteolysis by HIV-1 protease, and site-directed mutagenesis of this peptide in galactokinase revealed that the Glu residue at the P2' was important to binding to HIV-1 protease. Crystallographic analysis of HIV-1 protease complexed with a tight-binding peptide analogue inhibitor derived from this decapeptide sequence revealed that the "strand-loop" structure of the protein substrate must adopt a beta-sheet structure upon binding to the protease. The Glu residue in the P2' position of the inhibitor likely forms hydrogen-bonding interactions with both the alpha-amide and gamma-carboxylic groups of Asp-30 in the substrate binding site.     

The structure of a Michaelis serpin-protease complex.

Serine protease inhibitors (serpins) regulate the activities of circulating proteases. Serpins inhibit proteases by acylating the serine hydroxyl at their active sites. Before deacylation and complete proteolysis of the serpin can occur, massive conformational changes are triggered in the serpin while maintaining the covalent linkage between the protease and serpin. Here we report the structure of a serpin-trypsin Michaelis complex, which we visualized by using the S195A trypsin mutant to prevent covalent complex formation. This encounter complex reveals a more extensive interaction surface than that present in small inhibitor-protease complexes and is a template for modeling other serpin-protease pairs. Mutations of several serpin residues at the interface reduced the inhibitory activity of the serpin. The serine residue C-terminal to the scissile peptide bond is found in a closer than usual interaction with His 57 at the active site of trypsin.     

Catalytic and structural role of a metal-free histidine residue in bovine Cu-Zn superoxide dismutase

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.     

Computer modelling studies of ribonuclease T1-2'-deoxy-2'-fluoroguanylyl- (3',5')-cytidine complex.

The mode of binding of the substrate analog 2'-deoxy-2'-fluoroguanylyl- (3',5')-cytidine (GfpC) to RNase T1 was determined by computer modelling studies. The results obtained are in good agreement with the observations of 1H-nmr studies. The modes of binding of the substrate analog GfpC and the substrate GpC to the enzyme RNase T1 have been compared. Though the guanine base favours to occupy the same site of the enzyme in both the complexes, significant differences are observed in the local environment around the 2'-substituent group of guanosine ribose moiety. In the RNase T1-GpC complex, the 2'-OH group is in close proximity to the side chain carboxylic acid of Glu58 which leads to the formation of a hydrogen bond. However, in the RNase T1-GfpC complex, 2'-fluorine is positioned away from Glu58 due to electrostatic repulsion and instead forms a hydrogen bond with His40 imidazolium group. The results obtained rule out the possibility of His40 serving as the base group in catalysis as suggested by 1H-nmr studies and further support the primary role assigned to Glu58 as the general base group by earlier computer modelling and the recent site directed mutagenesis studies. This study also implies that the 2'-deoxy-2'-fluoro substrate analog may not serve as a good model for determining the amino acid residue which serves as the general base group in ribonuclease catalysed reactions.     

His92Ala mutation in ribonuclease T1 induces segmental flexibility. An X-ray study.

In the genetically mutated ribonuclease T1 His92Ala (RNase T1 His92Ala), deletion of the active site His92 imidazole leads to an inactive enzyme. Attempts to crystallize RNase T1 His92Ala under conditions used for wild-type enzyme failed, and a modified protocol produced two crystal forms, one obtained with polyethylene glycol (PEG), and the other with phosphate as precipitants. Space groups are identical to wild-type RNase T1, P2(1)2(1)2(1), but unit cell dimensions differ significantly, associated with different molecular packings in the crystals; they are a = 31.04 A, b = 62.31 A, c = 43.70 A for PEG-derived crystals and a = 32.76 A, b = 55.13 A, c = 43.29 A for phosphate-derived crystals, compared to a = 48.73 A, b = 46.39 A, c = 41.10 A for uncomplexed wild-type RNase T1. The crystal structures were solved by molecular replacement and refined by stereochemically restrained least-squares methods based on Fo greater than or equal to sigma (Fo) of 3712 reflections in the resolution range 10 to 2.2 A (R = 15.8%) for the PEG-derived crystal and based on Fo greater than or equal to sigma (Fo) of 6258 reflections in the resolution range 10 to 1.8 A (R = 14.8%) for the phosphate-derived crystal. The His92Ala mutation deletes the hydrogen bond His92N epsilon H ... O Asn99 of wild-type RNase T1, thereby inducing structural flexibility and conformational changes in the loop 91 to 101 which is located at the periphery of the globular enzyme. This loop is stabilized in the wild-type protein by two beta-turns of which only one is retained in the crystals obtained with PEG. In the crystals grown with phosphate as precipitant, both beta-turns are deleted and the segment Gly94-Ala95-Ser96-Gly97 is so disordered that it is not seen at all. In addition, the geometry of the guanine binding site in both mutant studies is different from "empty" wild-type RNase T1 but similar to that found in complexes with guanosine derivatives: the Glu46 side-chain carboxylate hydrogen bonds to Tyr42 O eta; water molecules that are present in the guanine binding site of "empty" wild-type RNase T1 are displaced; the Asn43-Asn44 peptide is flipped such that phi/psi-angles of Asn44 are in alpha L-conformation (that is observed in wild-type enzyme when guanine is bound).(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of type 1 ribonuclease H from hyperthermophilic archaeon Sulfolobus tokodaii: role of arginine 118 and C-terminal anchoring

The crystal structure of ribonuclease HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI) was determined at 1.6 A resolution. Sto-RNase HI exhibits not only RNase H activity but also double-stranded RNA-dependent ribonuclease (dsRNase) activity. The main-chain fold and steric configurations of the four acidic active-site residues of Sto-RNase HI are very similar to those of other type 1 RNases H. However, Arg118 of Sto-RNase HI is located at the position in which His124 of E. coli RNase HI, His539 of HIV-1 RNase H, and Glu188 of Bacillus halodurans RNase H are located. The mutation of this residue to Ala considerably reduced both the RNase H and dsRNase activities without seriously affecting substrate binding, suggesting that Arg118 is involved in catalytic function. This residue may promote product release by perturbing the coordination of the metal ion A as proposed for Glu188 of B. halodurans RNase H. In addition, the extreme C-terminal region of Sto-RNase HI is anchored to its core region by one disulfide bond and several hydrogen bonds. Differential scanning calorimetry measurements indicated that Sto-RNase HI is a hyperstable protein with a melting temperature of 102 degrees C. The mutations of the cysteine residues forming disulfide bond or elimination of the extreme C-terminal region greatly destabilized the protein, indicating that anchoring of the C-terminal tail is responsible for hyperstabilization of Sto-RNase HI.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

The Conserved Residue Arg46 in the N-Terminal Heptad Repeat Domain of HIV-1 gp41 Is Critical for Viral Fusion and Entry

During the process of HIV-1 fusion with the target cell, the N-terminal heptad repeat (NHR) of gp41 interacts with the C-terminal heptad repeat (CHR) to form fusogenic six-helix bundle (6-HB) core. We previously identified a crucial residue for 6-HB formation and virus entry - Lys63 (K63) in the C-terminal region of NHR (aa 54-70), which forms a hydrophobic cavity. It can form an important salt bridge with Asp121 (D121) in gp41 CHR. Here, we found another important conserved residue for virus fusion and entry, Arg46 (R46), in the N-terminal region of NHR (aa 35-53), which forms a hydrogen bond with a polar residue, Asn43 (N43), in NHR, as a part of the hydrogen-bond network. R46 can also form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HB's stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors.     

A norovirus protease structure provides insights into active and substrate binding site integrity

Norovirus 3C-like proteases are crucial to proteolytic processing of norovirus polyproteins. We determined the crystal structure of the 3C-like protease from Chiba virus, a norovirus, at 2.8-A resolution. An active site including Cys139 and His30 is present, as is a hydrogen bond network that stabilizes the active site conformation. In the oxyanion hole backbone, a structural difference was observed probably upon substrate binding. A peptide substrate/enzyme model shows that several interactions between the two components are critical for substrate binding and that the S1 and S2 sites appropriately accommodate the substrate P1 and P2 residues, respectively. Knowledge of the structure and a previous mutagenesis study allow us to correlate proteolysis and structure.     

Structural and enzymatic analysis of soybean beta-amylase mutants with increased pH optimum

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.     

Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated N?2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.     

Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.     

Helix-loop-helix peptide foldamers and their use in the construction of hydrolase mimetics

De novo designed helix-loop-helix peptide foldamers containing cis-2-aminocyclopentanecarboxylic acid residues were evaluated for their conformational stability and possible use in enzyme mimetic development. The correlation between hydrogen bond network size and conformational stability was demonstrated through CD and NMR spectroscopies. Molecules incorporating a Cys/His/Glu triad exhibited enzyme-like hydrolytic activity.