Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site

The specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the P1, P3, and P4 positions of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr↓Pro-Ile-Val-Gln) in human immunodeficiency virus type 1 (HIV-1). Previously, the substrate specificity of the P2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the family Retroviridae (P. Bagossi, T. Sperka, A. Fehér, J. Kádas, G. Zahuczky, G. Miklóssy, P. Boross, and J. Tözsér, J. Virol. 79:4213-4218, 2005). Our enzyme set comprised the proteases of HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus (AMV), Mason-Pfizer monkey virus, mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, human T-lymphotropic virus type 1, bovine leukemia virus, walleye dermal sarcoma virus, and human foamy virus. Molecular models were used to interpret the similarities and differences in specificity between these retroviral proteases. The results showed that the retroviral proteases had similar preferences (Phe and Tyr) for the P1 position in this sequence context, but differences were found for the P3 and P4 positions. Importantly, the sizes of the P3 and P4 residues appear to be a major contributor for specificity. The substrate specificities correlated well with the phylogenetic tree of the retroviruses. Furthermore, while the specificities of some enzymes belonging to different genera appeared to be very similar (e.g., those of AMV and MMTV), the specificities of the primate lentiviral proteases substantially differed from that observed for a nonprimate lentiviral protease.     

Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases.     

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.     

The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences

A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.     

Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid

Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.     

Closing of the flaps of HIV-1 protease induced by substrate binding: a model of a flap closing mechanism in retroviral aspartic proteases

The active site of aspartic proteases is covered by one or more flaps, which control access to the active site and play a significant role in the binding of the substrate. An extensive conformational change of the flaps takes place upon binding of substrate to the active site. A long molecular dynamics simulation was performed on the complex consisting of a peptide (CA-p2) from a natural substrate cleavage site of the gag/pol polyprotein placed in the active site of HIV-1 protease (PR) with an open flap conformation. During the simulation, the substrate induced the closing of the flaps into the closed conformation in an asymmetrical way through a hydrophobic intermediate state cluster. The nature of the residues of HIV-1 PR identified to be important in the flap closing mechanism is conserved across known structures of retroviral aspartic proteases family. The flap closing mechanism described in HIV-1 PR is proposed to be a general model for flap closing in retroviral aspartic proteases.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Specificity of serine proteases for cleavage sites on proatrial natriuretic factor

An immunological approach was used to investigate the specificity of protease cleavage sites on proANF. Cleavage of ³⁵S-cysteine biosynthetically-labeled proANF by whole serum, thrombin and kallikrein was examined. Reaction products were immunoprecipitated with two antibodies directed to different epitopes: a previously characterized antibody directed toward the carboxy-terminus of ANF_103–126, which cross reacts with proANF, ANF_99–126 and ANF_103–126, and a newly prepared antisera to synthetic ANF_99–105, which uniquely recognizes ANF_99–126, but not proANF or ANF_103–126. With increasing time of incubation with rat serum, proANF is sequentially cleaved at the C-terminus of a monobasic Pro-Arg dipeptide sequence to form ANF_99–126, and then at the C-terminus of a dibasic Arg-Arg dipeptide sequence to yield ANF_103–126. This cleavage activity of serum is blocked by leupeptin (40 μg/ml), but not by hirudin (100 nM), a specific inhibitor of thrombin, or by aprotinin (200 KIU/ml), a kallikrein inhibitor. When 100-fold purified serum cleavage enzyme was used in place of crude serum, similar results were obtained. Thrombin cleaves proANF only at the monobasic site to produce ANF_99–126 while kallikrein cleaves only at the dibasic site to produce ANF_103–126. As expected, the generation of these cleavage products can be inhibited by hirudin or aprotinin respectively. These data indicate that the substrate specificity of the serum cleavage activity is broader than that of thrombin or kallikrein, and that cleavage of proANF by serum proteases may be influenced by conformational restraints. The methods developed here should help in the future characterization of the physiological proANF cleaving enzyme.     

Amino acid insertions near Gag cleavage sites restore the otherwise compromised replication of human immunodeficiency virus type 1 variants resistant to protease inhibitors

A variety of amino acid substitutions in the protease and Gag proteins have been reported to contribute to the development of human immunodeficiency virus type 1 (HIV-1) resistance to protease inhibitors. In the present study, full-length molecular infectious HIV-1 clones were generated by using HIV-1 variants isolated from heavily drug-experienced and therapy-failed AIDS patients. Of six full-length infectious clones generated, four were found to have unique insertions (TGNS, SQVN, AQQA, SRPE, APP, and/or PTAPPA) near the p17/p24 and p1/p6 Gag cleavage sites, in addition to the known resistance-related multiple amino acid substitutions within the protease. The addition of such Gag inserts mostly compromised the replication of wild-type HIV-1, whereas the primary multidrug-resistant HIV infectious clones containing inserts replicated significantly better than those modified to lack the inserts. Western blot analyses revealed that the processing of Gag proteins by wild-type protease was impaired by the presence of the inserts, whereas that by mutant protease was substantially improved. The present study represents the first report clearly demonstrating that the inserts seen in the proximity of the Gag cleavage sites in highly multi-PI resistant HIV-1 variants restore the otherwise compromised enzymatic activity of mutant protease, enabling the multi-PI-resistant HIV-1 variants to remain replication competent.     

Amino acid residues involved in substrate binding and catalysis in an insect digestive beta-glycosidase

A beta-glycosidase (M(r) 50000) from Spodoptera frugiperda larval midgut was purified, cloned and sequenced. It is active on aryl and alkyl beta-glucosides and cellodextrins that are all hydrolyzed at the same active site, as inferred from experiments of competition between substrates. Enzyme activity is dependent on two ionizable groups (pK(a1)=4.9 and pK(a2)=7.5). Effect of pH on carbodiimide inactivation indicates that the pK(a) 7.5 group is a carboxyl. k(cat) and K(m) values were obtained for different p-nitrophenyl beta-glycosides and K(i) values were determined for a range of alkyl beta-glucosides and cellodextrins, revealing that the aglycone site has three subsites. Binding data, sequence alignments and literature beta-glycosidase 3D data supported the following conclusions: (1) the groups involved in catalysis were E(187) (proton donor) and E(399) (nucleophile); (2) the glycone moiety is stabilized in the transition state by a hydrophobic region around the C-6 hydroxyl and by hydrogen bonds with the other equatorial hydroxyls; (3) the aglycone site is a cleft made up of hydrophobic amino acids with a polar amino acid only at its first (+1) subsite.     

Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.     

Factor Xa subsite mapping by proteome-derived peptide libraries improved using WebPICS, a resource for proteomic identification of cleavage sites

Proteomic identification of protease cleavage site specificity (PICS) is a recent proteomic approach for the easy mapping of protease subsite preferences that determines both the prime- and non-prime side specificity concurrently. Here we greatly facilitate user access by providing an automated and simple web-based data-analysis resource termed WebPics (http://clipserve.clip.ubc.ca/pics/). We demonstrate the utility of WebPics analysis of PICS data by determining the substrate specificity of factor Xa from P6-P6', an important blood coagulation protease that proteolytically generates thrombin from prothrombin. PICS confirms existing data on non-prime site specificity and refines our knowledge of factor Xa prime-site selectivity.     

Unspecific arginine kinase of molecular weight 150 000. Amino acid composition, subunit structure and number of substrate binding sites.

The amino acid composition of unspecific arginine kinase of molecular weight 150 000 of Sabella pavonina muscle has been determined. If was found to be very similar to that of the phosphagen kinases previously studied. The subunit structure of the enzyme has been investigated by physical and chemical means. The data obtained from ultracentrifugation studies in 6 M guanidine hydrochloride and from molecular sieving and disc electrophoresis in 8 M urea, as well as the tryptic peptide mapping, suggest that Sabella muscle kinase is composed of four non-covalently linked polypeptide chains, with similar molecular weights. The number of binding sites for the nucleotide substrate ADP-Mg2+ has been estimated, using differential spectrophotometry and gel filtration on Sephadex columns. By both methods it was demonstrated that the enzyme contains two catalytic sites per protein molecule of molecular weight 150 000. Thus, arginine kinase from Sabella muscle, of molecular weight 150 000, consists of four similar polypeptide chains, but possesses only two substrate binding sites per tetrameric molecule.     

Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain

The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.     

Amino acid sequences around exofacial proteolytic cleavage sites of band 3 from bovine and porcine erythrocytes

• 1.1. Amino acid sequences of bovine and porcine band 3, an erythrocyte anion transporter, were determined.
• 2.2. The sequence of bovine band 3 was positioned to residues 519–599 (the numbering is based on human band 3), in which probably 6 residues were unidentified.
• 3.3. Binding site of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate), a potent anion transport inhibitor, was identified as Lys-539 in the bovine case.
• 4.4. A loop (residues 551–567), which provides exofacial proteolytic cleavage sites, contains only 53% homology between human and bovine, whereas the residues flanking it on either side are >84% homologous.
• 5.5. Furthermore, the loop of porcine band 3 was indicated to consist of a 6 or 7-residues short peptide as compared with those of other species.

     

Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.