Design of peptidase‐resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell‐permeant peptide Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys (PIK, P eptide I nhibitor of K inase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L ‐PIK in a biological milieu prompts for development of more stable L ‐PIK analogues for use as experimental tools in basic and drug‐oriented biomedical research. Previously, we designed PIK1, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys‐NH₂, that was 2.5‐fold more resistant to peptidases in human plasma in vitro than L ‐PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site‐protected peptides based on L ‐PIK and PIK1 degradation patterns in human plasma as revealed by ¹H‐NMR analysis. Implemented modifications increased half‐live of the PIK‐related peptides in plasma about 10‐fold, and these compounds retained 25–100% of L ‐PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐D ‐Arg‐Lys‐NH₂, was identified as the most stable and effective L ‐PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin‐induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell‐permeant inhibitors of MLCK in cell culture‐based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Site-directed mutagenesis of arginine 103 and lysine 185 in the proposed glycosaminoglycan-binding site of heparin cofactor II

Inhibition of thrombin by heparin cofactor (HCII) is accelerated approximately 1000-fold by heparin or dermatan sulfate. We found recently that the mutation Arg189----His decreases the affinity of HCII for dermatan sulfate but not for heparin (Blinder, M. A., Andersson, T. R., Abildgaard, U., and Tollefsen, D. M. (1989) J. Biol. Chem. 264, 5128-5133). Other investigators have implicated Arg47 and Lys125 of anti-thrombin (homologous to Arg103 and Lys185 of HCII) in heparin binding. To investigate the corresponding residues in HCII, we have constructed amino acid substitutions (Arg103----Leu, Gln, or Trp; Lys185----Met, Asn, or Thr) by oligonucleotide-directed mutagenesis of the cDNA and expressed the products in Escherichia coli. The recombinant HCII variants were assayed for binding to heparin-Sepharose and for inhibition of thrombin in the presence of various concentrations of heparin or dermatan sulfate. All of the Arg103 variants bound to heparin with normal affinity. Furthermore, inhibition of thrombin by the Arg103----Leu variant occurred at a normal rate in the absence of a glycosaminoglycan and was accelerated by normal concentrations of heparin and dermatan sulfate. These results indicate that HCII, unlike anti-thrombin, does not require a positive charge at this position for the interaction with heparin or dermatan sulfate. The Arg103----Gln and Arg103----Trp variants inhibited thrombin at about one-third of the normal rate in the absence of a glycosaminoglycan, suggesting that these mutations exert an effect on the reactive site (Leu444-Ser445) of HCII. All of the Lys185 variants bound to heparin with decreased affinity but inhibited thrombin at approximately the normal rate in the absence of a glycosaminoglycan. These variants required greater than 10-fold higher concentrations of heparin to accelerate inhibition of thrombin and were not stimulated significantly by dermatan sulfate, suggesting that heparin and dermatan sulfate interact with Lys185 of HCII. These results provide evidence that the glycosaminoglycan-binding site in HCII includes Lys185 but not Arg103, both of which were predicted to be involved by homology to anti-thrombin.     

Toward understanding the cationicity of defensins. Arg and Lys versus their noncoded analogs

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in alpha-defensins but not in beta-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human alpha-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six alpha-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys ((diMe)Lys), and homo-Arg ((homo)Arg). In addition, we prepared human beta-defensin 1 (hBD1) and (Lys-->Arg)hBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and (Lys-->Arg)hBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the alpha-defensin than in the beta-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.     

Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues.

Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase alpha-subunit sequence, were identified. Replacement of Thr286 with Ala (CaMK-(281-302 Ala286)) had no effect on either the potency (IC50 = 2 MicroM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala286) identified His282, Arg283, Lys291, Arg297, and Lys298 as important determinants (greater than 10-fold increase in IC50) for potent inhibition of CaMK-II. Glu285, Asp288, Lys291, Arg296, and Lys300 were not as essential (less than 4-fold change in IC50) for potent CaMK-II inhibition. Replacement of either Arg283, Lys291, or Arg297, and Lys298 with Ala did not alter the ATP-competitive mechanism of inhibition although the Ki values increased 16-530-fold. However, replacement of His282 with Ala decreased the IC50 by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His282 was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC50 of CaMK-(281-302, Ala286) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp286 mutant CaMK-II ((D286)mCaMK alpha) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D286)mCaMK alpha (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg283, Lys291, Arg297, and Lys298 are important for potent inhibition of CaMK-II and that the non-protonated form of His282 may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.     

Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Arginine 25 and Arginine 28 of lactoferrin are critical for effective heparin neutralization in blood

The interaction of lactoferrin with endogenous heparin-like molecules modulates glycosaminoglycan-mediated biological processes. We performed site-specific mutagenesis and expressed recombinant lactoferrin and lactoferrin mutants by the baculovirus insect cell expression system. Five basic residues at the lactoferrin N terminus; Arg 5, Arg 25, Arg 28, Lys 29, and Arg 31, were individually replaced by alanines. Heparin chromatography on fast-performance liquid chromatography system showed that the NaCl concentrations corresponding to the peak of each eluted recombinant protein from the column were 665, 620, 540, 550, 630, or 650 mM for wild-type recombinant lactoferrin, Arg 5, Arg 25, Arg 28, Lys 29, or Arg 31 recombinant lactoferrin mutant, respectively. We compared the ability of each mutated lactoferrin derivative to neutralize glycosaminoglycans in the thrombin serpin inhibition assays. In comparison to wild-type recombinant lactoferrin, all the mutants showed decreased ability to neutralize glycosaminoglycan in a dose-dependent manner. The mutations of lactoferrin at Arg 25 and Arg 28 demonstrated the most striking decrease in lactoferrin's ability to neutralize various glycosaminoglycans in both enzymatic and plasma clotting-based experiments. Therefore, our results suggest that Arg 25 and Arg 28 are the critical basic residues at the lactoferrin N terminus responsible for heparin binding. The other basic residues on the N terminus, Arg 5, Lys 29, and Arg 31, also contribute to heparin binding by presenting an additional cationic motif.     

A positively charged surface patch is important for hainantoxin‐IV binding to voltage‐gated sodium channels

Hainantoxin‐IV (HNTX‐IV), isolated from the venom of the spider Ornithoctonus hainana, is a specific antagonist of tetrodotoxin‐sensitive (TTX‐S) voltage‐gated sodium channels in rat dorsal root ganglion (DRG) cells. It adopts an inhibitor cystine knot motif, and structural analysis revealed a positively charged patch consisting of Arg26, Lys27, His28, Arg29 and Lys32 distributed on its molecular surface. Our previous study demonstrated that Lys27 and Arg29 but not Arg26 were critical residues for HNTX‐IV binding to TTX‐S sodium channels. In the present study, we examined the roles of His28 and Lys32 in the interaction of HNTX‐IV with its target. Two mutants, HNTX‐IV‐H28D and HNTX‐IV‐K32A, were generated by solid‐phase chemical synthesis and purified by reverse‐phase HPLC after refolding and oxidation, yielding two compounds of high purity with monoisotopic masses of 3962.66 and 3927.70 Da, respectively, as determined by MALDI‐TOF mass spectrometry. This indicated the presence of six cysteine residues forming three disulfide bonds. Moreover, circular dichroism spectroscopy analysis demonstrated that the substitution of His28 or Lys32 did not affect the overall structure of HNTX‐IV. The inhibitory activity of HNTX‐IV‐H28D and HNTX‐IV‐K32A against TTX‐S sodium channels in rat DRG cells was analyzed by whole‐cell patch‐clamp technique. The IC₅₀ values for the mutants were 0.57 and 5.80 μM (17‐fold and 170‐fold lower than the activity of the native toxin), indicating that His28 and Lys32 may be important for the inhibitory activity of HNTX‐IV. Taken together, our results suggest that the positively charged patch might be the binding site for the interaction of HNTX‐IV with TTX‐S sodium channels. These findings might contribute to the elucidation of the structure and function relationship of HNTX‐IV. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial activity,bactericidal mechanism and LPS‐neutralizing activity of the cell‐penetrating peptide pVEC and its analogs

pVEC is a cell‐penetrating peptide derived from the murine vascular endothelial‐cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)‐neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4–16 μM) against Gram‐positive and Gram‐negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 μM. These peptides induced a near‐complete membrane depolarization (more than 80%) at 4 μM against Staphylococcus aureus and a significant dye leakage (35–70%) from bacterial membrane‐mimicking liposome at a concentration as low as 1 μM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog‐derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor‐α release in LPS‐stimulated mouse macrophage RAW264.7 cells at 10 to 50 μM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS‐neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Chromogenic substrates of bovine beta-trypsin: the influence of an amino acid residue in P1 position on their interaction with the enzyme

The Cucurbita maxima trypsin inhibitor CMTI-III molecule was used as a vehicle to design and synthesize a series of trypsin chromogenic substrates modified in position P1: Ac-Ala-Val-Abu-Pro-X-pNA, where X = Orn, Lys, Arg, Har, Arg(NO(2)), Cit, Hci, Phe(p-CN), Phe(p-NH(2)); pNA = p-nitroanilide. The most active compounds (as determined by specificity constant k(cat)/K(m)) were peptides with the Arg and Lys residues in the position discussed. Changes in the length and the decrease of the positive charge of the amino acid residue side chain in position P(1) resulted in the decrease or loss of the affinity towards bovine beta-trypsin. Among peptides containing amino acid residues with uncharged side chains in position P1, only one with p-cyano-l-Phe revealed activity. These results correspond well with trypsin inhibitory activity of CMTI-III analogues modified in the equivalent position, indicating the same type of interaction between position P1 of the substrate or inhibitor and S1 site specificity of trypsin.     

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional role of "N" (nucleotide) and "P" (phosphorylation) domain interactions in the sarcoplasmic reticulum (SERCA) ATPase

Experimental perturbations of the nucleotide site in the N domain of the SR Ca2+ ATPase were produced by chemical derivatization of Lys492 or/and Lys515, mutation of Arg560 to Ala, or addition of inactive nucleotide analogue (TNP-AMP). Selective labeling of either Lys492 or Lys515 produces strong inhibition of ATPase activity and phosphoenzyme intermediate formation by utilization of ATP, while AcP utilization and reverse ATPase phosphorylation by Pi are much less affected. Cross-linking of the two residues with DIDS, however, drastically inhibits utilization of both ATP and AcP, as well as of formation of phosphoenzyme intermediate by utilization of ATP, or reverse phosphorylation by Pi. Mutation of Arg560 to Ala produces strong inhibition of ATPase activity and enzyme phosphorylation by ATP but has a much lower effect on enzyme phosphorylation by Pi. TNP-AMP increases the ATPase activity at low concentrations (0.1-0.3 microM), but inhibits ATP, AcP, and Pi utilization at higher concentration (1-10 microM). Cross-linking with DIDS and TNP-AMP binding inhibits formation of the transition state analogue with orthovanadate. It is concluded that in addition to the binding pocket delimited by Lys 492 and Lys515, Arg560 sustains an important and direct role in nucleotide substrate stabilization. Furthermore, the effects of DIDS and TNP-AMP suggest that approximation of N (nucleotide) and P (phosphorylation) domains is required not only for delivery of nucleotide substrate, but also to favor enzyme phosphorylation by nucleotide and nonnucleotide substrates, in the presence and in the absence of Ca2+. Domain separation is then enhanced by secondary nucleotide binding to the phosphoenzyme, thereby favoring its hydrolytic cleavage.     

Identification of essential charged residues in transmembrane segments of the multidrug transporter MexB of Pseudomonas aeruginosa

The MexABM efflux pump exports structurally diverse xenobiotics, utilizing the proton electrochemical gradient to confer drug resistance on Pseudomonas aeruginosa. The MexB subunit traverses the inner membrane 12 times and has two, two, and one charged residues in putative transmembrane segments 2 (TMS-2), TMS-4, and TMS-10, respectively. All five residues were mutated, and MexB function was evaluated by determining the MICs of antibiotics and fluorescent dye efflux. Replacement of Lys342 with Ala, Arg, or Glu and Glu346 with Ala, Gln, or Asp in TMS-2 did not have a discernible effect. Ala, Asn, or Lys substitution for Asp407 in TMS-4, which is well conserved, led to loss of activity. Moreover, a mutant with Glu in place of Asp407 exhibited only marginal function, suggesting that the length of the side chain at this position is important. The only replacements for Asp408 in TMS-4 or Lys939 in TMS-10 that exhibited significant function were Glu and Arg, respectively, suggesting that the native charge at these positions is required. In addition, double neutral mutants or mutants in which the charged residues Asp407 and Lys939 or Asp408 and Lys939 were interchanged completely lost function. An Asp408-->Glu/Lys939-->Arg mutant retained significant activity, while an Asp407-->Glu/Lys939-->Arg mutant exhibited only marginal function. An Asp407-->Glu/Asp408-->Glu double mutant also lost activity, but significant function was restored by replacing Lys939 with Arg (Asp407-->Glu/Asp408-->Glu/Lys939-->Arg). Taken as a whole, the findings indicate that Asp407, Asp408, and Lys939 are functionally important and raise the possibility that Asp407, Asp408, and Lys939 may form a charge network between TMS-4 and TMS-10 that is important for proton translocation and/or energy coupling.     

The C1-C2 interface residue lysine 50 of pig kidney fructose-1, 6-bisphosphatase has a crucial role in the cooperative signal transmission of the AMP inhibition.

To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.     

Functional role of putative critical residues in Mycobacterium tuberculosis RNase P protein

RNase P is involved in processing the 5⿲ end of pre-tRNA molecules. Bacterial RNase P contains a catalytic RNA subunit and a protein subunit. In this study, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other bacterial RNase Ps. The residues investigated in the current study include the unique residues, Val27, Ala70, Arg72, Ala77, and Asp124, and also Phe23 and Arg93 which have been found to be important in the function of RNase P protein components of other bacteria. The selected residues were individually mutated either to those present in other bacterial RNase P protein components at respective positions or in some cases to alanine. The wild type and mutant M. tuberculosis RNase P proteins were expressed in E. coli, purified, used to reconstitute holoenzymes with wild type RNA component in vitro, and functionally characterized. The Phe23Ala and Arg93Ala mutants showed very poor catalytic activity when reconstituted with the RNA component. The catalytic activity of holoenzyme with Val27Phe, Ala70Lys, Arg72Leu and Arg72Ala was also significantly reduced, whereas with Ala77Phe and Asp124Ser the activity of holoenzyme was similar to that with the wild type protein. Although the mutants did not suffer from any binding defects, Val27Phe, Ala70Lys, Arg72Ala and Asp124Ser were less tolerant towards higher temperatures as compared to the wild type protein. The K_m of Val27Phe, Ala70Lys, Arg72Ala and Ala77Phe were >2-fold higher than that of the wild type, indicating the substituted residues to be involved in substrate interaction. The study demonstrates that residues Phe23, Val27 and Ala70 are involved in substrate interaction, while Arg72 and Arg93 interact with other residues within the protein to provide it a functional conformation.     

Specificity of the basic side chains of Lys114, Lys125, and Arg129 of antithrombin in heparin binding

The anticoagulant polysaccharide heparin binds and activates the plasma proteinase inhibitor antithrombin through a pentasaccharide sequence. Lys114, Lys125, and Arg129 are the three most important residues of the inhibitor for pentasaccharide binding. To elucidate to what extent another positively charged side chain can fulfill the role of each of these residues, we have mutated Lys114 and Lys125 to Arg and Arg129 to Lys. Lys114 could be reasonably well replaced with Arg with only an approximately 15-fold decrease in pentasaccharide affinity, in contrast to an approximately 10(5)-fold decrease caused by substitution with an noncharged amino acid of comparable size. However, a loss of approximately one ionic interaction on mutation to Arg indicates that the optimal configuration of the network of basic residues of antithrombin that together interact with the pentasaccharide requires a Lys in position 114. Replacement of Lys125 with Arg caused an even smaller, approximately 3-fold, decrease in pentasaccharide affinity, compared with that of approximately 400-fold caused by mutation to a neutral amino acid. An Arg in position 125 is thus essentially equivalent to the wild-type Lys in pentasaccharide binding. Substitution of Arg129 with Lys decreased the pentasaccharide affinity an appreciable approximately 100-fold, a loss approaching that of approximately 400-fold caused by substitution with a neutral amino acid. Arg is thus specifically required in position 129 for high-affinity pentasaccharide binding. This requirement is most likely due to the ability of Arg to interact with other residues of antithrombin, primarily, Glu414 and Thr44, in a manner that appropriately positions the Arg side chain for keeping the pentasaccharide anchored to the activated state of the inhibitor.     

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.     

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

The plasminogen-binding group A streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine residues

The migration of the human pathogen Streptococcus pyogenes (group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the plasminogen-binding group A streptococcal M protein (PAM)-related protein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 microM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (K(d) = 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys(96) and Lys(101) reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg(107) and His(108) to alanine. Furthermore, mutagenesis of Arg(107) and His(108) abolished plasminogen binding by Prp despite the presence of Lys(96) and Lys(101) in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.