Protein-protein interactions in actin-myosin binding and structural effects of R405Q mutation: a molecular dynamics study

Detailed residue-wise interactions involved in the binding of myosin to actin in the rigor conformation without nucleotides have been examined using molecular dynamics simulations of the chicken skeletal myosin head complexed with two actin monomers, based on the cryo-microscopic model of Holmes et al. (Nature 2003;425:423-427). The overall interaction is largely electrostatic in nature, because of the charged residues in the four loops surrounding the central primary binding site. The 50k/20k loop, disordered in crystal structures and in simulations of free myosin in solution, was found to be in a conformation stabilized with 1 - 2 internal salt bridges. The cardiomyopathy loop forms 2 - 3 interprotein salt bridges with actin monomers upon binding, whereas its Arg405 residue, the mutation site associated with the hypertrophic cardiomyopathy, forms a strong salt bridge with Glu605 in the neighboring helix away from actin in the actin-bound myosin. The myopathy loop of the R405Q mutant maintains a high degree of two-strand beta-sheet character when bound to actin with the corresponding salt bridges broken.     

Expression and function of the gonadotropin-releasing hormone receptor are dependent on a conserved apolar amino acid in the third intracellular loop

The coupling of agonist-activated heptahelical receptors to their cognate G proteins is often dependent on the amino-terminal region of the third intracellular loop. Like many G protein-coupled receptors, the gonadotropin-releasing hormone (GnRH) receptor contains an apolar amino acid in this region at a constant distance from conserved Pro and Tyr/Asn residues in the fifth transmembrane domain (TM V). An analysis of the role of this conserved residue (Leu(237)) in GnRH receptor function revealed that the binding affinities of the L237I and L237V mutant receptors were unchanged, but their abilities to mediate GnRH-induced inositol phosphate signaling, G protein coupling, and agonist-induced internalization were significantly impaired. Receptor expression at the cell surface was reduced by replacement of Leu(237) with Val, and abolished by replacement with Ala, Arg, or Asp residues. These results are consistent with molecular modeling of the TM V and VI regions of the GnRH receptor, which predicts that Leu(237) is caged by several apolar amino acids (Ile(233), Ile(234), and Val(240) in TM V, and Leu(262), Leu(265), and Val(269) in TM VI) to form a tight hydrophobic cluster. These findings indicate that the conserved apolar residue (Leu(237)) in the third intracellular loop is an important determinant of GnRH receptor expression and activation, and possibly that of other G protein-coupled receptors.     

Identification of a novel prohormone sorting signal-binding site on carboxypeptidase E, a regulated secretory pathway-sorting receptor

Sorting of the prohormone POMC to the regulated secretory pathway necessitates the binding of a sorting signal to a sorting receptor, identified as membrane carboxypeptidase E (CPE). The sorting signal, located at the N terminus of POMC consists of two acidic (Asp10, Glu14) and two hydrophobic (Leu11, Leu18) residues exposed on the surface of an amphipathic loop. In this study, molecular modeling of CPE predicted that the acidic residues in the POMC-sorting signal bind specifically to two basic residues, Arg255 and Lys260, present in a loop unique to CPE, compared with other carboxypeptidases. To test the model, these two residues on CPE were mutated to Ser or Ala, followed by baculovirus expression of the mutant CPEs in Sf9 cells. Sf9 cell membranes containing CPE mutants with either Arg255 or Lys260, or both residues substituted, showed no binding of [125I]N-POMC1-26 (which contains the POMC-sorting signal motif), proinsulin, or proenkephalin. In contrast, substitution of an Arg147 to Ala147 at a substrate-binding site, Arg259 to Ala259 and Ser202 to Pro202, in CPE did not affect the level of [125I]N-POMC1-26 binding when compared with-wild type CPE. Furthermore, mutation of the POMC-sorting signal motif (Asp10, Leu11, Glu14, Leu18) eliminated binding to wild-type CPE. These results indicate that the sorting signal of POMC, proinsulin, and proenkephalin specifically interacts with Arg255 and Lys260 at a novel binding site, independent of the active site on CPE.     

Stabilizing and destabilizing clusters in the hydrophobic core of long two-stranded alpha-helical coiled-coils

Detailed sequence analyses of the hydrophobic core residues of two long two-stranded alpha-helical coiled-coils that differ dramatically in sequence, function, and length were performed (tropomyosin of 284 residues and the coiled-coil domain of the myosin rod of 1086 residues). Three types of regions were present in the hydrophobic core of both proteins: stabilizing clusters and destabilizing clusters, defined as three or more consecutive core residues of either stabilizing (Leu, Ile, Val, Met, Phe, and Tyr) or destabilizing (Gly, Ala, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Arg, Lys, and Trp) residues, and intervening regions that consist of both stabilizing and destabilizing residues in the hydrophobic core but no clusters. Subsequently, we designed a series of two-stranded coiled-coils to determine what defines a destabilizing cluster and varied the length of the destabilizing cluster from 3 to 7 residues to determine the length effect of the destabilizing cluster on protein stability. The results showed a dramatic destabilization, caused by a single Leu to Ala substitution, on formation of a 3-residue destabilizing cluster (DeltaT(m) of 17-21 degrees C) regardless of the stability of the coiled-coil. Any further substitution of Leu to Ala that increased the size of the destabilizing cluster to 5 or 7 hydrophobic core residues in length had little effect on stability (DeltaT(m) of 1.4-2.8 degrees C). These results suggested that the contribution of Leu to protein stability is context-dependent on whether the hydrophobe is in a stabilizing cluster or its proximity to neighboring destabilizing and stabilizing clusters.     

Role of charged and hydrophobic residues in the oligomerization of the PYRIN domain of ASC

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.     

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.     

Increase in bioluminescence intensity of firefly luciferase using genetic modification

Firefly luciferase is widely used for enzymatic measurement of ATP, and its gene is used as a reporter for gene expression experiments. From our mutant library, we selected novel mutations in Photinus pyralis luciferase with higher luminescence intensity. These included mutations at Ile423, Asp436, and Leu530. Luciferase is structurally composed of a large N-terminal active site domain (residues 1-436), a flexible linker (residues 436-440) peptide, and a small C-terminal domain (residues 440-550) facing the N domain. Thus, the mutations are located at the junction of the N-terminal domain and the flexible linker, in the flexible linker peptide, and in the tip of the C-terminal domain, respectively. Substitution of Asp436 with a nonbulky amino acid such as Gly remarkably increased the substrate affinity for ATP and d-luciferin. Substitution of Ile423 with a hydrophobic amino acid such as Leu and that of Leu530 with a positively charged amino acid such as Arg increased the substrate affinity and the turnover rate. Combining these mutations, we obtained luciferases that generate more than 10-fold higher luminescence intensity than the wild-type enzyme.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Identification of amino acids essential for calmodulin binding and activation of smooth muscle myosin light chain kinase.

Smooth muscle myosin light chain kinase (smMLCK) is a Ca(2+)-calmodulin (CaM)-dependent enzyme that phosphorylates the 20-kDa light chains of myosin. In a previous study (Bagchi, I.C., Kemp, B.E., and Means, A.R. (1989) J. Biol. Chem. 264, 15843-15849), we expressed in bacteria a 40-kDa fragment of smMLCK that displayed Ca(2+)-CaM-regulated catalytic activity. Initial mutagenesis experiments indicated that Gly811 and Arg812 were important for CaM-dependent activation of this 40-kDa enzyme. We have now carried out site-directed mutagenesis within the CaM-binding domain (Ser787 to Leu813) of this enzyme to identify amino acids that are critical for CaM binding and activation. Our studies reveal that the individual mutation of several hydrophobic amino acid residues such as Leu813, Ile810, and Trp800 and the glycine residue Gly804 also resulted in a severe decrease in or complete loss of CaM binding and activation of smMLCK. The hydrophobic residue (Trp800) and the basic residue (Arg812), both of which are mandatory for CaM binding to smMLCK, occur in analogous positions within the CaM-binding domain of a number of CaM-regulated enzymes. We conclude from these results that CaM binding by smMLCK is determined by an interplay of specific hydrophobic and electrostatic interactions which appear to be conserved among various target enzymes of CaM.     

Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H(+) for one extracellular Na(+). It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.     

Residues in the longitudinal, hydrophobic strip-of-helix relate to terminations and crossings of alpha-helices.

An alpha-helix terminates when the virtual extension of its most hydrophobic, longitudinal strip containing Leu, Ile, Val, Phe, and Met lacks those residues. In each of 247 helices a template was fitted to maximize the mean hydrophobicity of positions forming a longitudinal strip-of-helix. The template was then extended into sequences beyond the ends of the helices. Leu, Ile, Val, Phe, and Met occurred in positions in the longitudinal strip-of-helix at an increased frequency (p less than 0.001), but in the first and second positions beyond either end of each true helix, they occurred at the same frequency as for their empirical distribution over all the proteins. Excesses of Asp and Glu were found in the N-terminal loop, and of Arg, His, and Lys in specific positions about the C terminus of helices. The longitudinal hydrophobic strip, the smallest amino acid in that strip, and charged amino acids in that strip, related to rotational and longitudinal orientation of alpha-helices in 15 proteins. Adjacent helices generally crossed through their longitudinal hydrophobic strips. They usually crossed through the smallest residue in the strip. Charged residues, when they occurred in the strips, were excluded from the crossing regions.     

Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants.

To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket.

The S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site-directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA-Ala-Xaa-OH (Xaa = Leu, Glu, Lys, or Arg) and FA-Phe-Xaa-OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two- to three-fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D + L272D was found to have a preference for substrates with C-terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C-terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C-terminal amino acid sequence determinations.     

Roles of the transducin alpha-subunit alpha4-helix/alpha4-beta6 loop in the receptor and effector interactions

The visual GTP-binding protein, transducin, couples light-activated rhodopsin (R*) with the effector enzyme, cGMP phosphodiesterase in vertebrate photoreceptor cells. The region corresponding to the alpha4-helix and alpha4-beta6 loop of the transducin alpha-subunit (Gtalpha) has been implicated in interactions with the receptor and the effector. Ala-scanning mutagenesis of the alpha4-beta6 region has been carried out to elucidate residues critical for the functions of transducin. The mutational analysis supports the role of the alpha4-beta6 loop in the R*-Gtalpha interface and suggests that the Gtalpha residues Arg310 and Asp311 are involved in the interaction with R*. These residues are likely to contribute to the specificity of the R* recognition. Contrary to the evidence previously obtained with synthetic peptides of Gtalpha, our data indicate that none of the alpha4-beta6 residues directly or significantly participate in the interaction with and activation of phosphodiesterase. However, Ile299, Phe303, and Leu306 form a network of interactions with the alpha3-helix of Gtalpha, which is critical for the ability of Gtalpha to undergo an activational conformational change. Thereby, Ile299, Phe303, and Leu306 play only an indirect role in the effector function of Gtalpha.     

Conservative mutation Met8 --> Leu affects the folding process and structural stability of squash trypsin inhibitor CMTI-I

Protein molecules can accommodate a large number of mutations without noticeable effects on their stability and folding kinetics. On the other hand, some mutations can have quite strong effects on protein conformational properties. Such mutations either destabilize secondary structures, e.g., alpha-helices, are incompatible with close packing of protein hydrophobic cores, or lead to disruption of some specific interactions such as disulfide cross links, salt bridges, hydrogen bonds, or aromatic-aromatic contacts. The Met8 --> Leu mutation in CMTI-I results in significant destabilization of the protein structure. This effect could hardly be expected since the mutation is highly conservative, and the side chain of residue 8 is situated on the protein surface. We show that the protein destabilization is caused by rearrangement of a hydrophobic cluster formed by side chains of residues 8, Ile6, and Leu17 that leads to partial breaking of a hydrogen bond formed by the amide group of Leu17 with water and to a reduction of a hydrophobic surface buried within the cluster. The mutation perturbs also the protein folding. In aerobic conditions the reduced wild-type protein folds effectively into its native structure, whereas more then 75% of the mutant molecules are trapped in various misfolded species. The main conclusion of this work is that conservative mutations of hydrophobic residues can destabilize a protein structure even if these residues are situated on the protein surface and partially accessible to water. Structural rearrangement of small hydrophobic clusters formed by such residues can lead to local changes in protein hydration, and consequently, can affect considerably protein stability and folding process.     

Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect

Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F- actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin''s ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.     

Stabilization of the actomyosin complex by negative charges on myosin

Sequence comparisons of members of the myosin superfamily show a high degree of charge conservation in a surface exposed helix (Dictyostelium discoideum myosin II heavy chain residues S510 to K546). Most myosins display a triplet of acidic residues at the equivalent positions to D. discoideummyosin II residues D530, E531, and Q532. The high degree of charge conservation suggests strong evolutionary constrain and that this region is important for myosin function. Mutations at position E531 were shown to strongly affect actin binding [Giese, K. C., and Spudich, J. A. (1997) Biochemistry 36, 8465-8473]. Here, we used steady-state and transient kinetics to characterize the enzymatic competence of mutant constructs E531Q and Q532E, and their properties were compared with those of a loop 2 mutant with a 20 amino acid insertion containing 12 positive charges (20/+12) [Furch et al. (1998) Biochemistry 37, 6317-6326], double mutant Q532E(20/+12), and the native motor domain constructs. Our results confirm that charge changes at residues 531 and 532 primarily affect actin binding with little change being communicated to the nucleotide pocket. Mutation D531Q reduces actin affinity (K(A)) 10-fold, while Q532E leads to a 5-fold increase. The observed changes in K(A)() stem almost exclusively from variations in the dissociation rate constant (k(-A)), with the introduction of a single negative charge at position 532 having the same effect on k(-A) as the introduction of 12 positive charges in the loop 2 region.