Identification of residues at the alpha and epsilon subunit interfaces mediating species selectivity of Waglerin-1 for nicotinic acetylcholine receptors.

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the alpha-epsilon than to the alpha-delta binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the alpha-epsilon interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse epsilon subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat epsilon) and 115 (Tyr in mouse, Ser in rat epsilon) converts Wtx-1 affinity for the alpha-epsilon interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both alpha and epsilon subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.     

Hydrophobic pairwise interactions stabilize alpha-conotoxin MI in the muscle acetylcholine receptor binding site

The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.     

Conotoxin MI inhibits the alpha-delta acetylcholine binding site of the Torpedo marmorata receptor

The muscle-type nicotinic receptor has two pharmacologically distinguishable acetylcholine binding sites at the alpha-gamma and alpha-delta subunit interfaces; alpha-conotoxins can bind them selectively. As reported, alpha-conotoxin MI has greater affinity for the site near the alpha-delta interface of the BC(3)H1 cell receptor but, in the case of the Torpedo californica receptor, displays greater affinity for that near the alpha-gamma interface. To further investigate ligand selectivity, we study the conotoxin MI-Torpedo marmorata receptor interaction. In this work, we show the binding of alpha-conotoxin MI to the T. marmorata receptor and the influence of the antagonist alpha-Bungarotoxin and the agonist carbamylcholine on such binding; in addition, and contrasting with the results for the Torpedo californica receptor, we identify the alpha-delta subunit interface as the high affinity binding site. This is the first work describing different characteristics of the interaction between alpha-conotoxin MI and receptors from different species of the same genus.     

Pairwise interactions between neuronal alpha7 acetylcholine receptors and alpha-conotoxin ImI.

The present work uses alpha-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal alpha7 acetylcholine receptors. We identify key residues in alpha7 that contribute to CTx ImI affinity, and use mutant cycles analysis to identify pairs of residues that stabilize the receptor-conotoxin complex. We first mutated key residues in the seven known loops of alpha7 that converge at the subunit interface to form the ligand binding site. The mutant subunits were expressed in 293 HEK cells, and CTx ImI binding was measured by competition against the initial rate of 125I-alpha-bungarotoxin binding. The results reveal a predominant contribution by Tyr-195 in alpha7, accompanied by smaller contributions by Thr-77, Tyr-93, Asn-111, Gln-117, and Trp-149. Based upon our previous identification of bioactive residues in CTx ImI, we measured binding of receptor and toxin mutations and analyzed the results using thermodynamic mutant cycles. The results reveal a single dominant interaction between Arg-7 of CTx ImI and Tyr-195 of alpha7 that anchors the toxin to the binding site. We also find multiple weak interactions between Asp-5 of CTx ImI and Trp-149, Tyr-151, and Gly-153 of alpha7, and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of alpha7. The overall results establish the orientation of CTx ImI as it bridges the subunit interface and demonstrate close approach of residues on opposing faces of the alpha7 binding site.     

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.     

Contributions of Torpedo nicotinic acetylcholine receptor gamma Trp-55 and delta Trp-57 to agonist and competitive antagonist function

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.     

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.     

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.     

Structure of the agonist-binding sites of the Torpedo nicotinic acetylcholine receptor: affinity-labeling and mutational analyses identify gamma Tyr-111/delta Arg-113 as antagonist affinity determinants.

Photoaffinity labeling of the Torpedo nicotinic acetylcholine receptor (nAChR) with [3H]d-tubocurarine (dTC) has identified a residue within the gamma-subunit which, along with the analogous residue in delta-subunit, confers selectivity in binding affinities between the two agonist sites for dTC and alpha-conotoxin (alpha Ctx) MI. nAChR gamma-subunit, isolated from nAChR-rich membranes photolabeled with [3H]dTC, was digested with Staphylococcus aureus V8 protease, and a 3H-labeled fragment was purified by reversed-phase high-performance liquid chromatography. Amino-terminal sequence analysis of this fragment identified 3H incorporation in gamma Tyr-111 and gamma Tyr-117 at about 5% and 1% of the efficiency of [3H]dTC photoincorporation at gamma Trp-55, the primary site of [3H]dTC photoincorporation within gamma-subunit [Chiara, D. C., and Cohen, J. B. (1997) J. Biol. Chem 272, 32940-32950]. The Torpedo nAChR delta-subunit residue corresponding to gamma Tyr-111 (delta Arg-113) contains a positive charge which could confer the lower binding affinity seen for some competitive antagonists at the alpha-delta agonist site. To test this hypothesis, we examined by voltage-clamp analysis and/or by [125I]alpha-bungarotoxin competition binding assays the interactions of acetylcholine (ACh), dTC, and alpha Ctx MI with nAChRs containing gamma Y111R or delta R113Y mutant subunits expressed in Xenopus oocytes. While these mutations affected neither ACh equilibrium binding affinity nor the concentration dependence of channel activation, the gamma Y111R mutation decreased by 10-fold dTC affinity and inhibition potency. Additionally, each mutation conferred a 1000-fold change in the equilibrium binding of alpha Ctx MI, with delta R113Y enhancing and gamma Y111R weakening affinity. Comparison of these results with previous results for mouse nAChR reveals that, while the same regions of gamma- (or delta-) subunit primary structure contribute to the agonist-binding sites, the particular amino acids that serve as antagonist affinity determinants are species-dependent.     

Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis

Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.     

Determinants of alpha-conotoxin BuIA selectivity on the nicotinic acetylcholine receptor beta subunit

Neuronal nicotinic acetylcholine receptors (nAChRs) are pentamers composed of alpha and beta subunits. Different molecular compositions of these subunits constitute various receptor subtypes that are implicated in the pathophysiology and/or treatment of several disease states but are difficult to distinguish pharmacologically. Alpha-conotoxins are a group of small, structurally defined peptides that may be used to molecularly dissect the nAChR-binding site. Heteromeric nAChRs generally contain either a beta2 or beta4 subunit in addition to an alpha subunit at the ligand-binding interface. Alpha-conotoxin BuIA kinetically distinguishes between beta2- and beta4-containing nAChRs, with long off times for the latter. Mutational studies were used to assess the influence of residues that line the putative acetylcholine-binding pocket but differ between beta2 and beta4 subunits. Residues Thr/Lys59, Val/Ile111, and Phe/Gln119 of the respective beta2 and beta4 subunits are critical to off-rate differences. Among these residues, Thr59 of nAChR beta2 may interfere with effective access to the binding site, whereas Lys59 may facilitate this binding.     

Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

A common site of the Fc receptor gamma subunit interacts with the unrelated immunoreceptors FcalphaRI and FcepsilonRI

The transmembrane (TM) region of the Fc receptor-gamma (FcRgamma) chain is responsible for the association of this ubiquitous signal transduction subunit with many immunoreceptor ligand binding chains, making FcRgamma key to a number of leukocyte activities in immunity and disease. Some receptors contain a TM arginine residue that interacts with Asp-11 of the FcRgamma subunit, but otherwise the molecular basis for the FcRgamma subunit interactions is largely unknown. This study reports residues in the TM region of the FcRgamma subunit are important for association with the high affinity IgE receptor FcepsilonRI and a leukocyte receptor cluster member, the IgA receptor FcalphaRI. FcRgamma residue Leu-21 was essential for surface expression of FcepsilonRIalpha/gamma2 and Tyr-8, Leu-14, and Phe-15 contributed to expression. Likewise, detergent-stable FcRgamma association with FcalphaRI was also dependent on Leu-14 and Leu-21 and in addition required residues Tyr-17, Tyr-25, and Cys-26. Modeling the TM regions of the FcRgamma dimer indicated these residues interacting with both FcalphaRI and FcepsilonRI are near the interface between the two FcRgamma TM helices. Furthermore, the FcRgamma residues interacting with FcalphaRI form a leucine zipper-like interface with mutagenesis confirming a complementary interface comprising FcalphaRI residues Leu-217, Leu-220, and Leu-224. The dependence of these two nonhomologous receptor interactions on FcRgamma Leu-14 and Leu-21 suggests that all the associated Fc receptors and the activating leukocyte receptor cluster members interact with this one site. Taken together these data provide a molecular basis for understanding how disparate receptor families assemble with the FcRgamma subunit.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

The epsilon subunit and inhibitory monoclonal antibodies interact with the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase

The epitopes of two classes of monoclonal antibody and the binding site for the epsilon subunit have been mapped to the carboxyl-terminal region of the beta subunit of Escherichia coli F1-ATPase using partial CNBr cleavage, weak acid hydrolysis, and Western blots. One class of antibody, B-I, inhibits ATPase activity; the other class, B-II, recognizes an epitope not exposed on the surface of intact F1. Data from two-dimensional gels and blots of beta cleaved with CNBr/weak acid showed that the B-I epitope lies between Asp-381 and the carboxyl-terminal Leu-459, and the B-II epitope lies between Asp-345 and Met-380. Weak acid hydrolysis of the beta-epsilon product obtained by cross-linking F1 with a water-soluble carbodiimide yielded a fragment containing epsilon and a 13-kDa carboxyl-terminal fragment of beta indicating that epsilon interacts with this portion of beta as well. Fab fragments from the B-I antibody beta-6 could be cross-linked to the epsilon subunit in native F1 by various cross-linking agents demonstrating that the antibody and the epsilon subunit occupy adjacent, nonoverlapping sites on the beta subunit. Implications of these results for the roles of the epsilon subunit and of the carboxyl-terminal region of the beta subunit in F1 are discussed.     

Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167.

Vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes relaxation of supercoiled DNA by cleaving and rejoining DNA strands through a DNA- (3'-phosphotyrosyl)-enzyme intermediate. We have performed a kinetic analysis of mutational effects at four essential amino acids: Arg-130, Gly-132, Tyr-136 and Lys-167. Arg-130, Gly-132 and Lys-167 are conserved in all members of the type IB topoisomerase family. Tyr-136 is conserved in all poxvirus topoisomerases. We show that Arg-130 and Lys-167 are required for transesterification chemistry. Arg-130 enhances the rates of both cleavage and religation by 10(5). Lys-167 enhances the cleavage and religation reactions by 10(3) and 10(4), respectively. An instructive distinction between these two essential residues is that Arg-130 cannot be replaced by lysine, whereas substituting Lys-167 by arginine resulted in partial restoration of function relative to the alanine mutant. We propose that both basic residues interact directly with the scissile phosphate at the topoisomerase active site. Mutations at positions Gly-132 and Tyr-136 reduced the rate of strand cleavage by more than two orders of magnitude, but elicited only mild effects on religation rate. Gly-132 and Tyr-136 are suggested to facilitate a pre-cleavage activation step. The results of comprehensive mutagenesis of the vaccinia topoisomerase illuminate mechanistic and structural similarities to site-specific recombinases.     

Site-directed mutagenesis of stable adenosine triphosphate synthase

Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity.     

Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region.

A docking model of the alpha(2) I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of alpha(2) (residues 284-288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole alpha(2)beta(1), with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. alpha(1), alpha(2), and alpha(10)) bind to collagen in a similar fashion.