Macromolecular substrate affinity for the tissue factor-factor VIIa complex is independent of scissile bond docking.

The upstream coagulation enzymes are homologous trypsin-like serine proteases that typically function in enzyme-cofactor complexes, exemplified by coagulation factor VIIa (VIIa), which is allosterically activated upon binding to its cell surface receptor tissue factor (TF). TF cooperates with VIIa to create a bimolecular recognition surface that serves as an exosite for factor X binding. This study analyzes to what extent scissile bond docking to the catalytic cleft contributes to macromolecular substrate affinity. Mutation of the P1 Arg residue in factor X to Gln prevented activation by the TF.VIIa complex but did not reduce macromolecular substrate affinity for TF.VIIa. Similarly, mutations of the S and S' subsites in the catalytic cleft of the enzyme VIIa failed to reduce affinity for factor X, although the affinity for small chromogenic substrates and the efficiency of factor X scissile bond cleavage were reduced. Thus, docking of the activation peptide bond to the catalytic cleft of this enzyme-cofactor complex does not significantly contribute to affinity for macromolecular substrate. Rather, it appears that the creation of an extended macromolecular substrate recognition surface involving enzyme and cofactor is utilized to generate substrate specificity between the highly homologous, regulatory proteases of the coagulation cascade.     

Lysine residues 165 and 166 are essential for the cofactor function of tissue factor

Tissue factor, a 45-kilodalton membrane glycoprotein, is an essential cofactor for the plasma serine protease factor VII which activates factor X in the first step of the extrinsic coagulation cascade. Two adjacent lysine residues (numbers 165 and 166) were identified in the extracytoplasmic domain of tissue factor that are crucial for function. Site-directed mutagenesis of both lysines to alanines results in complete loss of activity. Mutation of either lysine alone results in a molecule which is much more sensitive to the phospholipid composition of the activating surface than the wild-type molecule. It is postulated that interactions between the extracytoplasmic domain of tissue factor and the membrane surface are necessary for bound factor VII or VIIa to assume a conformation capable of efficient catalysis.     

Identification of residues in the N-terminal domain of the Yersinia tyrosine phosphatase that are critical for substrate recognition

YopH is a 468-amino acid protein-tyrosine phosphatase that is produced by pathogenic Yersinia species. YopH is translocated into host mammalian cells via a type III protein secretion system. Translocation of YopH into human epithelial cells results in dephosphorylation of p130(Cas) and paxillin, disruption of focal adhesions, and inhibition of integrin-mediated bacterial phagocytosis. Previous studies have shown that the N-terminal 129 amino acids of YopH comprise a bifunctional domain. This domain binds to the SycH chaperone in Yersinia to orchestrate translocation and to tyrosine-phosphorylated target proteins in host cells to mediate substrate recognition. We used random mutagenesis in combination with the yeast two-hybrid system to identify residues in the YopH N-terminal domain that are involved in substrate-binding activity. Four single codon changes (Q11R, V31G, A33D, and N34D) were identified that interfered with binding of the YopH N-terminal domain to tyrosine-phosphorylated p130(Cas) but not to SycH. These mutations did not impair YopH translocation into HeLa cells infected with Yersinia pseudotuberculosis. Introduction of the V31G substitution into catalytically inactive (substrate-trapping) forms of YopH interfered with the ability of these proteins to bind to p130(Cas) and to localize to focal adhesions in HeLa cells. In addition, the V31G substitution reduced the ability of catalytically active YopH to dephosphorylate target proteins in HeLa cells. These data indicate that the substrate- and SycH-binding activities of the YopH N-terminal domain can be separated and that the former activity is important for recognition and dephosphorylation of substrates by YopH in vivo.     

Identification of critical residues for G-1 addition and substrate recognition by tRNA(His) guanylyltransferase

The yeast tRNA(His) guanylyltransferase (Thg1) is an essential enzyme in yeast. Thg1 adds a single G residue to the 5' end of tRNA(His) (G(-1)), which serves as a crucial determinant for aminoacylation of tRNA(His). Thg1 is the only known gene product that catalyzes the 3'-5' addition of a single nucleotide via a normal phosphodiester bond, and since there is no identifiable sequence similarity between Thg1 and any other known enzyme family, the mechanism by which Thg1 catalyzes this unique reaction remains unclear. We have altered 29 highly conserved Thg1 residues to alanine, and using three assays to assess Thg1 catalytic activity and substrate specificity, we have demonstrated that the vast majority of these highly conserved residues (24/29) affect Thg1 function in some measurable way. We have identified 12 Thg1 residues that are critical for G(-1) addition, based on significantly decreased ability to add G(-1) to tRNA(His) in vitro and significant defects in complementation of a thg1Delta yeast strain. We have also identified a single Thg1 alteration (D68A) that causes a dramatic decrease in the rigorous specificity of Thg1 for tRNA(His). This single alteration enhances the k(cat)/K(M) for ppp-tRNA(Phe) by nearly 100-fold relative to that of wild-type Thg1. These results suggest that Thg1 substrate recognition is at least in part mediated by preventing recognition of incorrect substrates for nucleotide addition.     

A critical role of lysine residues in the stimulation of tissue plasminogen activator by denatured proteins and fibrin clots

Two-chain 70 000-dalton plasminogen activator of tissue origin displays only weak activity toward plasminogen in a two-component system. The rate of activation is enhanced a minimum of 50-fold by the presence of fibrin clots or denatured proteins. The stimulation must depend on both chemical determinants and spatial configuration, since native proteins, including fibrinogen, lack significant stimulatory activity. These studies employed chemical modifications of four stimulatory proteins (fibrin, denatured fibrinogen, denatured IgG and denatured ovalbumin) to identify a critical role for lysine residues. Arginine, aspartic acid, cysteine, cystine, glutamic acid, histidine, methionine, tyrosine and tryptophan were found not to be essential. The critical spatial determinant(s) remain(s) unknown.     

Specificity in substrate binding by protein folding catalysts: tyrosine and tryptophan residues are the recognition motifs for the binding of peptides to the pancreas-specific protein disulfide isomerase PDIp

Using a cross-linking approach, we recently demonstrated that radiolabeled peptides or misfolded proteins specifically interact in vitro with two luminal proteins in crude extracts from pancreas microsomes. The proteins were the folding catalysts protein disulfide isomerase (PDI) and PDIp, a glycosylated, PDI-related protein, expressed exclusively in the pancreas. In this study, we explore the specificity of these proteins in binding peptides and related ligands and show that tyrosine and tryptophan residues in peptides are the recognition motifs for their binding by PDIp. This peptide-binding specificity may reflect the selectivity of PDIp in binding regions of unfolded polypeptide during catalysis of protein folding.     

The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity

The ubiquitous ribonucleases (RNases) play important roles in RNA metabolism, angiogenesis, neurotoxicity, and antitumor or antimicrobial activity. Only the antimicrobial RNases possess high positively charged residues, although their mechanisms of action remain unclear. Here, we report on the role of cationic residues of human RNase7 (hRNase7) in its antimicrobial activity. It exerted antimicrobial activity against bacteria and yeast, even at 4 degrees C. The bacterial membrane became permeable to the DNA-binding dye SYTOX(R) Green in only a few minutes after bactericidal RNase treatment. NMR studies showed that the 22 positively charged residues (Lys(18) and Arg(4)) are distributed into three clusters on the surface of hRNase7. The first cluster, K(1),K(3),K(111),K(112), was located at the flexible coil near the N terminus, whereas the other two, K(32),K(35) and K(96),R(97),K(100), were located on rigid secondary structures. Mutagenesis studies showed that the flexible cluster K(1),K(3),K(111),K(112), rather than the catalytic residues His(15), Lys(38), and His(123) or other clusters such as K(32),K(35) and K(96),R(97),K(100), is critical for the bactericidal activity. We suggest that the hRNase7 binds to bacterial membrane and renders the membrane permeable through the flexible and clustered Lys residues K(1),K(3),K(111),K(112). The conformation of hRNase7 can be adapted for pore formation or disruption of bacterial membrane even at 4 degrees C.     

Amino acid residues that are critical for in vivo catalytic activity of CtpA, the carboxyl-terminal processing protease for the D1 protein of photosystem II.

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.     

A critical epitope for substrate recognition by the nucleosome remodeling ATPase ISWI

The ATPase ISWI is the catalytic core of several nucleosome remodeling complexes, which are able to alter histone–DNA interactions within nucleosomes such that the sliding of histone octamers on DNA is facilitated. Dynamic nucleosome repositioning may be involved in the assembly of chromatin with regularly spaced nucleosomes and accessible regulatory sequence elements. The mechanism that underlies nucleosome sliding is largely unresolved. We recently discovered that the N-terminal ‘tail’ of histone H4 is critical for nucleosome remodeling by ISWI. If deleted, nucleosomes are no longer recognized as substrates and do not stimulate the ATPase activity of ISWI. We show here that the H4 tail is part of a more complex recognition epitope which is destroyed by grafting the H4 N-terminus onto other histones. We mapped the H4 tail requirement to a hydrophilic patch consisting of the amino acids R₁₇H₁₈R₁₉ localized at the base of the tail. These residues have been shown earlier to contact nucleosomal DNA, suggesting that ISWI recognizes an ‘epitope’ consisting of the DNA-bound H4 tail. Consistent with this hypothesis, the ISWI ATPase is stimulated by isolated H4 tail peptides ISWI only in the presence of DNA. Acetylation of the adjacent K₁₂ and K₁₆ residues impairs substrate recognition by ISWI.     

Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.     

C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.     

Identification of factor Xa residues critical for interaction with protein Z-dependent protease inhibitor: both active site and exosite interactions are required for inhibition

Protein Z-dependent protease inhibitor (ZPI) is a plasma serpin, which can rapidly inactivate factor Xa (fXa) in the presence of protein Z (PZ), negatively charged phospholipids, and Ca2+. To investigate the mechanism by which ZPI inactivates fXa, we expressed the serpin in mammalian cells and characterized its reactivity with both wild-type and selected mutants of fXa that 1) contained substitutions in the autolysis loop and the heparin binding exosite, 2) lacked the first EGF-like domain (fXa-des-EGF-1), or 3) contained the Gla domain of protein C (fXa/PC-Gla). Inhibition studies in both the presence and absence of PZ revealed that Arg-143, Lys-147, and Arg-154 of the autolysis loop and Lys-96, Lys-169, and Lys-236 of the heparin binding exosite are required for recognition of ZPI, with Arg-143 being essential for the interaction. Similar studies with fXa-des-EGF-1 and fXa/PC-Gla suggested that protein-protein interaction with either the Gla or the EGF-1 domain may not play a dominant role in the PZ-dependent recognition of fXa by the serpin on phospholipid vesicles. Further studies showed that an inactive Ser-195 to Ala mutant of fXa effectively competes with wild-type fXa for binding to the non-serpin inhibitors tissue factor pathway inhibitor and recombinant tick anticoagulant peptide, but does not compete for binding to ZPI. This suggests that the catalytic residue of fXa is required for interaction with ZPI.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.     

Hydrophilic residues 526 KNDAAD 531 in the flexible C-terminal region of the chaperonin GroEL are critical for substrate protein folding within the central cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.     

Arginine residues are critical for the heparin-cofactor activity of antithrombin III.

A dilution/quench technique was used to monitor the time course of chemical modification on the heparin-cofactor (a) and progressive thrombin-inhibitory (b) activities of human antithrombin III. Treatment of antithrombin III (AT III) with 2,4,6-trinitrobenzenesulphonate at pH 8.3 and 25 degrees C leads to the loss of (a) at 60-fold more rapid rate than the loss of (b). This is consistent with previous reports [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Pecon & Blackburn (1984) J. Biol. Chem. 259, 935-938] that lysine residues are involved in the binding of heparin to AT III, but not in thrombin binding. Treatment of AT III with phenylglyoxal at pH 8.3 and 25 degrees C again leads to a more rapid loss of (a) than of (b), with the loss of the former proceeding at a 4-fold faster rate. The presence of heparin during modification with phenylglyoxal significantly decreases the rate of loss of (a). Full loss of (a) correlates with the modification of seven arginine residues per inhibitor molecule, whereas loss of (b) does not commence until approximately four arginine residues are modified and is complete upon the modification of approximately eleven arginine residues per inhibitor molecule. This suggests that (the) arginine residue(s) in AT III are involved in the binding of heparin in addition to the known role of Arg-393 at the thrombin-recognition site [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Jörnvall, Fish & Björk (1979) FEBS Lett. 106, 358-362].     

Probing the interface between factor Xa and tissue factor in the quaternary complex tissue factor-factor VIIa-factor Xa-tissue factor pathway inhibitor.

Blood coagulation is triggered by the formation of a complex between factor VIIa (FVIIa) and its cofactor, tissue factor (TF). TF-FVIIa is inhibited by tissue factor pathway inhibitor (TFPI) in two steps: first TFPI is bound to the active site of factor Xa (FXa), and subsequently FXa-TFPI exerts feedback inhibition of TF-FVIIa. The FXa-dependent inhibition of TF-FVIIa activity by TFPI leads to formation of the quaternary complex TF-FVIIa-FXa-TFPI. We used site-directed fluorescence probing to map part of the region of soluble TF (sTF) that interacts with FXa in sTF-FVIIa-FXa-TFPI. We found that the C-terminal region of sTF, including positions 163, 166, 200 and 201, is involved in binding to FXa in the complex, and FXa, most likely via its Gla domain, is also in contact with the Gla domain of FVIIa in this part of the binding region. Furthermore, a region that includes the N-terminal part of the TF2 domain and the C-terminal part of the TF1 domain, i.e. the residues 104 and 197, participates in the interaction with FXa in the quaternary complex. Moreover, comparisons of the interaction areas between sTF and FX(a) in the quaternary complex sTF-FVIIa-FXa-TFPI and in the ternary complexes sTF-FVII-FXa or sTF-FVIIa-FX demonstrated large similarities.     

An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease.

Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1. It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed. These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1. In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction. Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well. These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin.     

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.     

Six specific lysine residues are crucial in maintaining the structure and function of soluble manganese stabilizing protein

When manganese stabilizing protein （MSP） was treated with 0.5 mM N-succinimidyl propionate （NSP）, the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered, in spite of changes of MSP surface Lys residues. Furthermore, far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondary and tertiary structure. Mapping of the sites of NSP modification by Staphylococcus V8 protease digestion of the modified protein, as well as analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry, indicated that seven Lys residues were modified. The results suggested that these residues are not absolutely essential to the structure and function of MSP. However, when the NSP concentration was increased to 4 mM, the modified MSP was unable to bind photosystem Ⅱ and completely lost its reactivating capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clear conformational change in MSP after 4 mM NSP treatment, suggesting that some Lys residues are involved in maintaining the structure and function of MSP. Analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that another six Lys residues, namely Lys20, Lys 101, Lys196, Lys207, Lysl30 （or Lys137） and Lys66 （or Lys76）, were modified by 4 mM NSP. Therefore, these six Lys residues are crucial in maintaining the structure and function of soluble MSP.