Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of "effective charge balance"

In thymidylate synthase, four conserved arginines provide two hydrogen bonds each to the oxygens of the phosphate group of the substrate, 2'-deoxyuridine-5'-monophosphate. Of these, R23, R178, and R179 are far removed from the site of methyl transfer and contribute to catalysis solely through binding and orientation of ligands. These arginines can be substituted by other residues, while still retaining more than 1% activity of the wild-type enzyme. We compared the kinetics and determined the crystal structures of dUMP complexes of three of the most active, uncharged single mutants of these arginines, R23I, R178T, and R179T, and of double mutants (R23I, R179T) and (R178T, R179T). The dramatically higher K(m) for R178T compared to the other two single mutants arises from the effects of R178 substitution on the orientation of dUMP; 10-15-fold increases in for R23I and R178T reflect the role of these residues in stabilizing the closed conformation of TS in ternary complexes. The free energy for productive dUMP binding, DeltaG(S), increases by at least 1 kcal/mol for each mutant, even when dUMP orientation and mobility in the crystal structure is the same as in wild-type enzyme. Thus, the four arginines do not contribute excess positive charge to the PO(4)(-2) binding site; rather, they ideally complement the charge and geometry of the phosphate moiety. More-than-additive increases in DeltaG(S) seen in the double mutants are consistent with quadratic increases in DeltaG(S) predicted for deviations from ideal electrostatic interactions and may also reflect cooperative binding of the arginines to the phosphate oxygens.     

Decreased agonist affinity and chloride conductance of mutant glycine receptors associated with human hereditary hyperekplexia.

Hereditary hyperekplexia is a dominant neurological disorder associated with point mutations at the channel-forming segment M2 of the glycine receptor alpha 1 subunit. Voltage-clamp recordings from the heterologously expressed mutants (alpha 1R271L or alpha 1R271Q) revealed 146- to 183-fold decreased potencies of glycine to activate the chloride channel, and significantly reduced maximal whole-cell currents as compared with wild-type receptors. In contrast, the ability of the competitive antagonist strychnine to block glycine-induced currents was similar in all cases. Radioligand binding assays showed a 90- to 1365-fold reduction in the ability of glycine to displace [3H]strychnine from its binding site on the mutant receptors. Paralleling the reductions in whole-cell current, the elementary main-state conductances of the mutants (alpha 1R271L, 64 pS; alpha 1R271Q, 14 pS) were lower than that of the wild-type receptor (86 pS). The decreased agonist affinities and chloride conductances of the mutants are likely to cause neural hyperexcitability of affected patients by impairing glycinergic inhibition. In addition, our data reveal that structural modifications of the ion-channel region can affect agonist binding to the glycine receptor.     

A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.     

Transition state stabilization by six arginines clustered in the active site of creatine kinase

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.     

Mutation underlying resistance of Plasmodium berghei to atovaquone in the quinone binding domain 2 (Qo(2)) of the cytochrome b gene

The anti-malarial agent atovaquone specifically targets the cytochrome bc₁ complex and inhibits the parasite respiration. Resistance to this drug, a coenzyme Q analogue, is associated with mutations in the mitochondrial cytochrome b gene. We previously reported atovaquone resistant mutations in Plasmodium berghei, in the first quinone binding domain (Qo₁) of the cytochrome b gene (M133I and L144S) with V284F in the sixth transmembrane domain. However, in P. falciparum the most common mutations are found in the Qo₂ region. To obtain a better model for biochemical and genetic studies, we have now extended our study to isolate a wider range of P. berghei resistant strains, in particular those in the Qo₂. Here we report four new mutations (Y268N, Y268C, L271V and K272R), all in the Qo₂ domain. Two of these mutations are convergent to codon 268 (nt802–804) drug-induced mutation in P. falciparum.     

MASP-1 Induced Clotting – The First Model of Prothrombin Activation by MASP-1

Mannan-binding lectin-associated serine protease-1 (MASP-1), a protein of the complement lectin pathway, resembles thrombin in terms of structural features and substrate specificity. Due to its interplay with several coagulation factors, it has the ability to induce fibrin clot formation independent of the usual coagulation activation pathways. We have recently shown that MASP-1 activates prothrombin and identified arginine (R) 155, R271, and R393 as potential cleavage sites. FXa cleaves R320 instead of R393, and thrombin cleaves R155 and R284 in prothrombin. Here we have used three arginine-to-glutamine mutants of prothrombin, R271Q, R320Q, R393Q and the serine-to-alanine active site mutant S525A to investigate in detail the mechanism of MASP-1 mediated prothrombin activation. Prothrombin wildtype and mutants were digested with MASP-1 and the cleavage products were analysed by SDS-PAGE and N-terminal sequencing. A functional clotting assay was performed by thrombelastography. We have found that MASP-1 activates prothrombin via two simultaneous pathways, either cleaving at R271 or R393 first. Both pathways result in the formation of several active alternative thrombin species. Functional studies confirmed that both R393 and R320 are required for prothrombin activation by MASP-1, whereas R155 is not considered to be an important cleavage site in this process. In conclusion, we have described for the first time a detailed model of prothrombin activation by MASP-1.     

The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4.

Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.     

Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor

Central core disease (CCD) is a human myopathy that involves a dysregulation in muscle Ca(2)+ homeostasis caused by mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), the protein that comprises the calcium release channel of the SR. Although genetic studies have clearly demonstrated linkage between mutations in RyR1 and CCD, the impact of these mutations on release channel function and excitation-contraction coupling in skeletal muscle is unknown. Toward this goal, we have engineered the different CCD mutations found in the NH(2)-terminal region of RyR1 into a rabbit RyR1 cDNA (R164C, I404M, Y523S, R2163H, and R2435H) and characterized the functional effects of these mutations after expression in myotubes derived from RyR1-knockout (dyspedic) mice. Resting Ca(2)+ levels were elevated in dyspedic myotubes expressing four of these mutants (Y523S > R2163H > R2435H R164C > I404M RyR1). A similar rank order was also found for the degree of SR Ca(2)+ depletion assessed using maximal concentrations of caffeine (10 mM) or cyclopiazonic acid (CPA, 30 microM). Although all of the CCD mutants fully restored L-current density, voltage-gated SR Ca(2)+ release was smaller and activated at more negative potentials for myotubes expressing the NH(2)-terminal CCD mutations. The shift in the voltage dependence of SR Ca(2)+ release correlated strongly with changes in resting Ca(2)+, SR Ca(2)+ store depletion, and peak voltage-gated release, indicating that increased release channel activity at negative membrane potentials promotes SR Ca(2)+ leak. Coexpression of wild-type and Y523S RyR1 proteins in dyspedic myotubes resulted in release channels that exhibited an intermediate degree of SR Ca(2)+ leak. These results demonstrate that the NH(2)-terminal CCD mutants enhance release channel sensitivity to activation by voltage in a manner that leads to increased SR Ca(2)+ leak, store depletion, and a reduction in voltage-gated Ca(2)+ release. Two fundamentally distinct cellular mechanisms (leaky channels and EC uncoupling) are proposed to explain how altered release channel function caused by different mutations in RyR1 could result in muscle weakness in CCD.     

Probing the role of arginines and histidines in the catalytic function and activation of yeast 3-phosphoglycerate kinase by site-directed mutagenesis

A cluster of conserved histidines and arginines (His-62, His-167, Arg-21, Arg-38, and Arg-168) in 3-phosphoglycerate kinase (PGK) has been implicated as possibly involved in the binding of 3-phosphoglycerate (3-PG) and/or stabilization of the negatively charged transition state. The role of these residues in the catalytic function of yeast PGK and in the substrate- and sulfate-dependent activation was investigated by site-directed mutagenesis. The following substitutions, R21A, R21Q, H62Q, H167S, and R168Q, produced functional enzymes. In contrast, the R38A and R38Q mutations resulted in a complete loss of catalytic activity. These results demonstrate that of the basic residues studied, only arginine 38 is essential for the catalytic function of PGK. A moderate decrease in the catalytic efficiency as the result of the R21A, H167S, and R168Q mutations and an increased catalytic efficiency of the H62Q mutant rule out a possible role of a positive charge at these positions in the mechanism of phosphoryl transfer reaction. In contrast to the wild type PGK and the H62Q mutant, both of which are activated at low and inhibited at high sulfate concentration, the H167S, R168Q, and R21A mutants exhibited a progressive inhibition with increased concentration of sulfate. The activation observed at high concentration of either ATP or 3-PG as a variable substrate in the steady-state kinetics of wild type PGK was abolished as the result of the latter three mutations. The results of this work support the hypothesis that PGK has two binding sites for anionic ligands, the catalytic and regulatory sites for each substrate and the activatory and inhibitory sites for sulfate, and suggest that arginine 21, arginine 168, and histidine 167 are located in the activatory anion binding site, common for sulfate, 3-PG, and ATP. The increased Km values for both substrates and decreased specific activities of the mutants suggest that this regulatory site is close to the catalytic site.     

Role of charged residues at the OmpF porin channel constriction probed by mutagenesis and simulation

The channel constriction of OmpF porin, a pore protein in the bacterial outer membrane, is highly charged due to the presence of three arginines (R42, R82, and R132) and two acidic residues (D113 and E117). The influence of these charges on ion conductance, ion selectivity, and voltage gating has been studied with mutants D113N/E117Q, R42A/R82A/R132A/D113N/E117Q, and V18K/G131K, which were designed to remove or add protein charge at the channel constriction. The crystal structures revealed no or only local changes compared to wild-type OmpF, thus allowing a comparative study. The single-channel conductance of the isosteric D113N/E117Q variant was found to be 2-fold reduced, and that of the pentuple mutant was 70% of the wild-type value, despite a considerably larger pore cross section. Ion selectivity was drastically altered by the mutations with cation/anion permeability ratios ranging from 1 to 12. Ion flow through these and eight other mutants, which have been characterized previously, was simulated by Brownian dynamics based on the detailed crystal structures. The calculated ion selectivity and relative channel conductance values agree well with the experimental data. This demonstrates that ion translocation through porin is mainly governed by pore geometry and charge, the two factors that are properly represented in the simulations.     

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.     

A Small Molecule Agonist THIQ as a Novel Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

Although mutations in the melanocortin-4 receptor (MC4R) gene cause severe early-onset obesity, we still do not have effective approaches to correct the defects of these mutations. Several antagonists have been identified as pharmacoperones of the MC4R whereas no agonist of the MC4R has been reported. In the present study, we investigated the effect of a small molecule agonist of the MC4R, THIQ, on the cell surface expression and signaling of ten intracellularly retained MC4R mutants using different cell lines. We showed that THIQ increased the cell surface expression of three mutants (N62S, C84R, and C271Y) and two of them (N62S and C84R) had increased signaling in HEK293 cells. Interestingly, THIQ increased the signaling of two other mutants (P78L and P260Q) without increasing their cell surface expression in HEK293 cells. In neuronal cells, THIQ exhibited a more potent effect, correcting the cell surface expression and signaling of seven mutants (N62S, I69R, P78L, C84R, W174C, P260Q, and C271Y). Other mutants were not rescued by THIQ. We also showed that THIQ did not rescue MC4R mutants defective in ligand binding or signaling or one intracellularly retained mutant of the melanocortin-3 receptor. In summary, we demonstrated that a small molecule agonist acted as a pharmacoperone of the MC4R rescuing the cell surface expression and signaling of some intracellularly retained MC4R mutants.     

Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor

The murine scavenger receptor class B, type I (mSR-BI) is a receptor for high density lipoprotein (HDL), low density lipoprotein (LDL), and acetylated LDL (AcLDL). It mediates selective uptake of lipoprotein lipid and stimulates efflux of [(3)H]cholesterol to lipoproteins. SR-BI-mediated [(3)H]cholesterol efflux was proposed to be independent of ligand binding. In this study, using anti-mSR-BI antibody KKB-1 and two mSR-BI mutants with altered ligand binding properties, we demonstrated that SR-BI-mediated [(3)H]cholesterol efflux to lipoproteins was correlated with ligand binding and lipid uptake activities of the receptor. The KKB-1 antibody, which blocked lipoprotein binding without substantially altering the cholesterol oxidase-accessible cellular [(3)H]cholesterol, also blocked [(3)H]cholesterol efflux to HDL and LDL. One of the SR-BI mutants, which has a double substitution of arginines for glutamines at positions 402 and 418 (Q402R/Q418R), exhibited a high level of LDL binding and lipid uptake from LDL, but lost most of the corresponding HDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to LDL, but not to HDL. Another mutant, M158R, with an arginine in place of methionine at position 158, exhibited reduced HDL and LDL receptor activities, but apparently normal AcLDL receptor activity. This mutant could mediate efficient [(3)H]cholesterol efflux to AcLDL, but not to HDL or LDL. These results suggest that SR-BI-stimulated [(3)H]cholesterol efflux to lipoproteins critically depends on ligand binding to this receptor and raise the possibility that the mechanisms of selective lipid uptake and [(3)H]cholesterol efflux may be intimately related.     

Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins

Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of "receptor discriminator" residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β(2)-adrenergic receptors (β(2)AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β(2)AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β(2)AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β(2)AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β(2)AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes.     

Calcium-binding sites of calmodulin and electron transfer by inducible nitric oxide synthase

Like that of the neuronal nitric oxide synthase (nNOS), the binding of Ca(2+)-bound calmodulin (CaM) also regulates the activity of the inducible isoform (iNOS). However, the role of each of the four Ca(2+)-binding sites of CaM in the activity of iNOS is unclear. Using a series of single-point mutants of Drosophila melanogaster CaM, the effect that mutating each of the Ca(2+)-binding sites plays in the transfer of electrons within iNOS has been examined. The same Glu (E) to Gln (Q) mutant series of CaM used previously [Stevens-Truss, R., Beckingham, K., and Marletta, M. A. (1997) Biochemistry 36, 12337-12345] to study the role of the Ca(2+)-binding sites in the activity of nNOS was used for these studies. We demonstrate here that activity of iNOS is dependent on Ca(2+) being bound to sites II (B2Q) and III (B3Q) of CaM. Nitric oxide ((*)NO) producing activity (as measured using the hemoglobin assay) of iNOS bound to the B2Q and B3Q CaMs was found to be 41 and 43% of the wild-type activity, respectively. The site I (B1Q) and site IV (B4Q) CaM mutants only minimally affected (*)NO production (95 and 90% of wild-type activity, respectively). These results suggest that NOS isoforms, although all possessing a prototypical CaM binding sequence and requiring CaM for activity, interact with CaM differently. Moreover, iNOS activation by CaM, like nNOS, is not dependent on Ca(2+) being bound to all four Ca(2+)-binding sites, but has specific and distinct requirements. This novel information, in addition to helping us understand NOS, should aid in our understanding of CaM target activation.     

Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.9 A resolution, but neither the ligand nor the sulfate from the crystallization buffer was found in the active site. A second structure determined with crystals prepared in the presence of dUMP and the antifolate 10-propargyl-5,8-dideazafolate revealed that the inhibitor was bound in an extended, nonproductive conformation, partially occupying the nucleotide-binding site. A sulfate ion, rather than dUMP, was found in the nucleotide phosphate-binding site. Previous studies have shown that the substitution at three of the four arginines of the dUMP phosphate-binding site is permissive; however; for Arg166, all the mutations lead to a near-inactive mutant. The present structures of TS R166Q reveal that the phosphate-binding site is largely intact, but with a substantially reduced affinity for phosphate, despite the presence of the three remaining arginines. The position of Cys146, which initiates catalysis, is shifted in the mutant and resides in a position that interferes with the binding of the dUMP pyrimidine moiety.     

Polygalacturonase-inhibiting protein interacts with pectin through a binding site formed by four clustered residues of arginine and lysine

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs.     

Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca2+ modulator matures human hair cuticle

S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.     

Molecular determinants of the binding specificity of BH3 ligands to BclXL apoptotic repressor

B‐cell lymphoma extra‐large protein (BclXL) serves as an apoptotic repressor by virtue of its ability to recognize and bind to BH3 domains found within a diverse array of proapoptotic regulators. Herein, we investigate the molecular basis of the specificity of the binding of proapoptotic BH3 ligands to BclXL. Our data reveal that while the BH3 ligands harboring the LXXX[A/S]D and [R/Q]XLXXXGD motif bind to BclXL with high affinity in the submicromolar range, those with the LXXXGD motif afford weak interactions. This suggests that the presence of a glycine at the fourth position (G+4)—relative to the N‐terminal leucine (L0) within the LXXXGD motif—mitigates binding, unless the LXXXGD motif also contains arginine/glutamine at the ?2 position. Of particular note is the observation that the residues at the +4 and ?2 positions within the LXXX[A/S]D and [R/Q]XLXXXGD motifs appear to be energetically coupled—replacement of either [A/S]+4 or [R/Q]‐2 with other residues has little bearing on the binding affinity of BH3 ligands harboring one of these motifs. Collectively, our study lends new molecular insights into understanding the binding specificity of BH3 ligands to BclXL with important consequences on the design of novel anticancer drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 573–582, 2014.