Structure/function analyses of human sex hormone-binding globulin: effects of zinc on steroid-binding specificity

In humans, sex hormone-binding globulin (SHBG) binds and transports the biologically most important androgens and estrogens in the blood, and regulates the access of these steroids to their targets tissues. In addition to binding sex steroids, SHBG has specific binding sites for divalent cations including calcium and zinc. Zinc binding to a site at the entrance of the steroid-binding pocket in human SHBG has been shown to reduce its affinity for estrogens, while having no impact on the binding of C19 steroids. Crystallographic studies indicate that C18 and C19 steroids are bound in opposite orientations within the SHBG steroid-binding site, and we have obtained new information that supports a molecular model explaining the mechanism by which zinc alters the affinity of human SHBG for estrogens, by studying directly the estradiol-binding properties SHBG variants created by site-directed mutagenesis. In this model, the coordination of a zinc ion by the side chains of residues Asp65 and His136 eliminates a critical hydrogen bond between Asp65 and the hydroxyl at C3 of estrogens, such as estradiol and 2-methoxyestradiol, and causes disorder in a polypeptide loop segment that covers the steroid-binding site. The combination of these structural changes explains the specific decrease in the affinity of human SHBG for C18 steroids in the presence of a zinc ion.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

Function of His185 in Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase

Aquifex aeolicus 3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) by favoring the activation of a water molecule coordinated to the active-site metal ion. Cys11, His185, Glu222 and Asp233 are the other metal ligands. Wild-type KDO8PS is purified with Zn(2+) or Fe(2+) in the active site, but maximal activity in vitro is achieved when the endogenous metal is replaced with Cd(2+). The H185G enzyme retains 8% of the wild-type activity. ICP mass spectrometry analysis indicates that loss of His185 decreases the enzyme affinity for Fe(2+), but not for Zn(2+). However, maximal activity is again achieved by substitution of the endogenous metal with Cd(2+). We have determined the X-ray structures of the Cd(2+) H185G enzyme in its substrate-free form, and in complex with PEP, and PEP plus A5P. These structures show a normal amount of Cd(2+) bound, suggesting that coordination by His185 is not essential to retain Cd(2+) in the active site. Nonetheless, there are significant changes in the coordination sphere of Cd(2+) with respect to the wild-type enzyme, as the carboxylate moiety of PEP binds directly to the metal ion and replaces water and His185 as ligands. These observations indicate that the primary function of His185 in A.aeolicus KDO8PS is to orient PEP in the active site of the enzyme in such a way that a water molecule on the sinister (si) side of PEP can be activated by direct coordination to the metal ion.     

The identification of cation-binding domains on the surface of microsomal cytochrome b5 using 1H-NMR paramagnetic difference spectroscopy.

One-dimensional and two-dimensional 1H-NMR methods and paramagnetic difference spectroscopy have defined cation binding domains on the surface of the tryptic fragment of microsomal cytochrome b5. The addition of tris(ethylenediamine) chromium(III) [Cr(en)3(3+)] to solutions of ferricytochrome b5 reveals at least three distinct sites on the surface of the protein to which highly charged cations may bind (20 mM phosphate pH 7.0, T = 300 K). Surprisingly only one of these sites is located close to the haem edge region of the protein, whilst the remaining two sites are more remote. Site I contains the exposed haem C13 propionate and a series of carboxylate residues that includes glutamates 37, 38, 43, 44, and 48. Sites II and III are located away from the haem edge region and are delineated by the broadening of aromatic resonances of histidines 26 and 80. Further investigation of the interaction between Cr(en)3(3+) and cytochrome b5 using two-dimensional double-quantum-filtered correlated spectroscopy shows that resonances assigned to Glu59, Asp60, Glu79, Asp82 and Asp83 are broadened with the distribution of these charged side chains correlating with the relaxation broadening observed from one-dimensional experiments. In a binary complex with ferricytochrome c, Cr(en3(3+) broadens many cytochrome b45 resonances including the haem propionates, His26, Ala54, Thr55 and His80. Although the pattern of line-broadening of resonances at sites II and III is unaltered by complex formation, cytochrome c shields residues at site I, the haem edge site. The results indicate that the interaction between cytochrome b5 and c in a binary complex involves multiple protein configurations.     

Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol

In a crystal structure of the amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG), the biologically active estrogen metabolite, 2-methoxyestradiol (2-MeOE2), binds in the same orientation as estradiol. The high affinity of SHBG for 2-MeOE2 relies primarily on hydrogen bonding between the hydroxyl at C-3 of 2-MeOE2 and Asp(65) and an interaction between the methoxy group at C-2 and the amido group of Asn(82). Accommodation of the 2-MeOE2 methoxy group causes an outward displacement of residues Ser(128)-Pro(130), which appears to disorder and displace the loop region (Leu(131)-His(136)) that covers the steroid-binding site. This could influence the binding kinetics of 2-MeOE2 and/or facilitate ligand-dependent interactions between SHBG and other proteins. Occupancy of a zinc-binding site reduces the affinity of SHBG for 2-MeOE2 and estradiol in the same way. The higher affinity of SHBG for estradiol derivatives with a halogen atom at C-2 is due to either enhanced hydrogen bonding between the hydroxyl at C-3 and Asp(65) (2-fluoroestradiol) or accommodation of the functional group at C-2 (2-bromoestradiol), rather than an interaction with Asn(82). By contrast, the low affinity of SHBG for 2-hydroxyestradiol can be attributed to intra-molecular hydrogen bonding between the hydroxyls in the aromatic steroid ring A, which generates a steric clash with the amido group of Asn(82). Understanding how C-2 derivatives of estradiol interact with SHBG could facilitate the design of biologically active synthetic estrogens.     

Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.     

Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features

alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase

Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).     

Altering of the metal specificity of Escherichia coli alkaline phosphatase

Analysis of sequence alignments of alkaline phosphatases revealed a correlation between metal specificity and certain amino acid side chains in the active site that are metal-binding ligands. The Zn(2+)-requiring Escherichia coli alkaline phosphatase has an Asp at position 153 and a Lys at position 328. Co(2+)-requiring alkaline phosphatases from Thermotoga maritima and Bacillus subtilis have a His and a Trp at these positions, respectively. The mutations D153H, K328W, and D153H/K328W were induced in E. coli alkaline phosphatase to determine whether these residues dictate the metal dependence of the enzyme. The wild-type and D153H enzymes showed very little activity in the presence of Co(2+), but the K328W and especially the D153H/K328W enzymes effectively use Co(2+) for catalysis. Isothermal titration calorimetry experiments showed that in all cases except for the D153H/K328W enzyme, a possible conformation change occurs upon binding Co(2+). These data together indicate that the active site of the D153H/K328W enzyme has been altered significantly enough to allow the enzyme to utilize Co(2+) for catalysis. These studies suggest that the active site residues His and Trp at the E. coli enzyme positions 153 and 328, respectively, at least partially dictate the metal specificity of alkaline phosphatase.     

Refinement of a homology model of the mu-opioid receptor using distance constraints from intrinsic and engineered zinc-binding sites

Publication of the rhodopsin X-ray structure has facilitated the development of homology models of other G protein-coupled receptors. However, possible shifts of transmembrane (TM) alpha helices, expected variations in helical distortions, and differences in loop size necessitate experimental verification of these comparative models. To refine a rhodopsin-based homology model of the mu-opioid receptor (MOR), we experimentally determined structural-distance constraints from intrinsic and engineered metal-binding sites in the rat MOR. Investigating the relatively high intrinsic affinity of MOR for Zn(2+) (IC(50) approximately 30microM), we observed that mutation of His(319) (TM7) abolished Zn(2+) inhibition of ligand binding, while mutation of Asp(216) (extracellular loop 2) decreased the effect of Zn(2+), suggesting these residues participate in the intrinsic Zn(2+)-binding center of MOR. To verify the relative orientation of TM5 and TM6 and to examine whether a rhodopsin-like alpha aneurism is present in TM5, we engineered Zn(2+)-binding centers by mutating residues of TM5 and TM6 to Cys or His, making use of the native His(297) in TM6 as an additional Zn(2+)-coordination site. Inhibition of opioid ligand binding by Zn(2+) suggests that residues Ile(234) and Phe(237) in TM5 face the binding-site crevice and form a metal-binding center with His(297) and Val(300) in TM6. This observation is inconsistent with a rhodopsin-like structure, which would locate Ile(234) on the lipid-exposed side of TM5, too distant from other residues making up the Zn(2+)-binding site. Subsequent distance geometry refinement of the MOR model indicates that the rhodopsin-like alpha aneurism is likely absent in TM2 but present in TM5.     

Ca2+ occlusion and gating function of Glu309 in the ADP-fluoroaluminate analog of the Ca2+-ATPase phosphoenzyme intermediate

In the absence of ATP the sarcoplasmic reticulum ATPase (SERCA) binds two Ca(2+) with high affinity. The two bound Ca(2+) rapidly undergo reverse dissociation upon addition of EGTA, but can be distinguished by isotopic exchange indicating fast exchange at a superficial site (site II), and retardation of exchange at a deeper site (site I) by occupancy of site II. Site II mutations that allow high affinity binding to site I, but only low affinity binding to site II, show that retardation of isotopic exchange requires higher Ca(2+) concentrations with the N796A mutant, and is not observed with the E309Q mutant even at millimolar Ca(2+). Fluoroaluminate forms a complex at the catalytic site yielding stable analogs of the phosphoenzyme intermediate, with properties similar to E2-P or E1-P.Ca(2). Mutational analysis indicates that Asp(351), Lys(352), Thr(353), Asp(703), Asn(706), Asp(707), Thr(625), and Lys(684) participate in stabilization of fluoroaluminate and Mg(2+) at the phosphorylation site. In the presence of fluoroaluminate and Ca(2+), ADP (or AMP-PCP) favors formation of a stable ADP.E1-P.Ca(2) analog. This produces strong occlusion of Ca(2+) bound to both sites (I and II), whereby dissociation occurs very slowly even following addition of EGTA. Occlusion by fluoraluminate and ADP is not observed with the E309Q mutant, suggesting a gating function of Glu(309) at the mouth of a binding cavity with a single path of entry. This phenomenon corresponds to the earliest step of the catalytic cycle following utilization of ATP. Experiments on limited proteolysis reveal that a long range conformational change, involving displacement of headpiece domains and transmembrane helices, plays a mechanistic role.     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Metal ion binding to human hemopexin

Binding of divalent metal ions to human hemopexin (Hx) purified by a new protocol has been characterized by metal ion affinity chromatography and potentiometric titration in the presence and absence of bound protoheme IX. ApoHx was retained by variously charged metal affinity chelate resins in the following order: Ni(2+) > Cu(2+) > Co(2+) > Zn(2+) > Mn(2+). The Hx-heme complex exhibited similar behavior except the order of retention of the complex on Zn(2+)- and Co(2+)-charged columns was reversed. One-dimensional (1)H NMR of apoHx in the presence of Ni(2+) implicates at least two His residues and possibly an Asp, Glu, or Met residue in Ni(2+) binding. Potentiometric titrations establish that apoHx possesses more than two metal ion binding sites and that the capacity and/or affinity for metal ion binding is diminished when heme binds. For most metal ions that have been studied, potentiometric data did not fit to binding isotherms that assume one or two independent binding sites. For Mn(2+), however, these data were consistent with a high-affinity site [K(A) = (15 +/- 3) x 10(6) M(-)(1)] and a low-affinity site (K(A) 相似文献   

Zinc(II)-mediated inhibition of ribonuclease Sa by an N-hydroxyurea nucleotide and its basis

Ribonuclease Sa (RNase Sa) is a secretory ribonuclease from Streptomyces aureofaciens. Herein, 3'-N-hydroxyurea-3'-deoxythymidine 5'-phosphate is shown to be a competitive inhibitor of catalysis by RNase Sa. Inhibition is enhanced by nearly 10-fold in the presence of Zn(2+), which could coordinate to the N-hydroxyurea group along with enzymic residues. The carboxylate of Glu54 is the putative base that abstracts a proton from the 2' hydroxyl group during catalysis of RNA cleavage by RNase Sa. Replacing Glu54 with a glutamine residue has no effect on the affinity of N-hydroxyurea 1 for the enzyme, but eliminates the zinc(II)-dependence of that affinity. These data indicate that an N-hydroxyurea nucleotide can recruit Zn(2+) to inhibit the enzymatic activity of RNase Sa, and suggest that the carboxylate of Glu54 is a ligand for that Zn(2+). These findings further the development of a new class of ribonuclease inhibitors based on the complex of an N-hydroxyurea nucleotide and zinc(II).     

Novel cytotoxic chelators that bind iron(II) selectively over zinc(II) under aqueous aerobic conditions

To achieve cellular iron deprivation by chelation, it is important to develop chelators with selective metal-binding properties. Selectivity for iron has long been the province of certain oxygen-donor chelators such as desferrioxamine, which target Fe(III) and exploit the strength of a relatively ionic Fe(III)-O interaction. We have been studying novel chelators that possess mechanisms to selectively chelate +2 biometals, particularly tachpyr [N,N',N"-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane] and derivatives from N,N',N"-trialkylation and pyridine ring alkylation. Metal-exchange and metal-binding competition reactions have been conducted at pH 7.4, 37 degrees C and time periods until no further change was observed (generally 24-48 h). Under anaerobic conditions, tachpyr is strongly selective for iron, binding 95+/-5% Fe(II) versus 5+/-5% Zn(II) in the forms [Fe(tachpyr)](2+) and [Zn(tachpyr)](2+) respectively. Under aerobic conditions, tachpyr complexes Fe(II) more effectively than Fe(III), forming iminopyridyl complexes [Fe(tachpyr-ox-n)](2+) (n=2, 4) by O(2)-induced and iron-mediated oxidative dehydrogenation. Complexes [Fe(tachpyr-ox-n)](2+) are also strongly bound forms of iron that are unaffected by an excess of Zn(II) (75 mol zinc:1 mol iron complex). The preference of tachpyr for iron over zinc under aerobic conditions appears to be hindered by oxidation of Fe(II) to Fe(III), such that the proportions bound are 44+/-10% Fe(II) versus 56+/-10% Zn(II), in the respective forms [Fe(tachpyr-ox-n)](2+) and [Zn(tachpyr)](2+). However, upon addition of the reducing agent Na(2)S(2)O(4) that converts Fe(III) to Fe(II), the binding proportions shift to 76+/-10% Fe(II) versus 24+/-10% Zn(II), demonstrating a clear preference of tachpyr for Fe(II) over Zn(II). Iron(II) is in the low-spin state in [Fe(tachpyr)](2+) and [Fe(tachpyr-ox-n)](2+) (n=2, 4), which is a likely cause of the observed selectivity. N-methylation of tachpyr [giving (N-methyl)(3)tachpyr] results in the loss of selectivity for Fe(II), which is attributed to the steric effect of the methyl groups and a resulting high-spin state of Fe(II) in [Fe(N-methyl)(3)tachpyr)](2+). The relationship of chelator selectivity to cytotoxicity in the tach family will be discussed.     

Tuning magnesium sensitivity of BK channels by mutations

Intracellular Mg(2+) at physiological concentrations activates mSlo1 BK channels by binding to a metal-binding site in the cytosolic domain. Previous studies suggest that residues E374, Q397, and E399 are important in Mg(2+) binding. In the present study, we show that mutations of E374 or E399 to other amino acids, except for Asp, abolish Mg(2+) sensitivity. These results further support that the side chains of E374 and E399 are essential for Mg(2+) coordination. To the contrary, none of the Q397 mutations abolishes Mg(2+) sensitivity, suggesting that its side chain may not coordinate to Mg(2+). However, because Q397 is spatially close to E374 and E399, its mutations affect the Mg(2+) sensitivity of channel gating by either reducing or increasing the Mg(2+) binding affinity. The pattern of mutational effects and the effect of chemical modification of Q397C indicate that Q397 is involved in the Mg(2+)-dependent activation of BK channels and that mutations of Q397 alter Mg(2+) sensitivity by affecting the conformation of the Mg(2+) binding site as well as by electrostatic interactions with the bound Mg(2+) ion.     

Crystal structures of fructose 1,6-bisphosphatase: mechanism of catalysis and allosteric inhibition revealed in product complexes

Crystal structures of metal-product complexes of fructose 1, 6-bisphosphatase (FBPase) reveal competition between AMP and divalent cations. In the presence of AMP, the Zn(2+)-product and Mg(2+)-product complexes have a divalent cation present only at one of three metal binding sites (site 1). The enzyme is in the T-state conformation with a disordered loop of residues 52-72 (loop 52-72). In the absence of AMP, the enzyme crystallizes in the R-state conformation, with loop 52-72 associated with the active site. In structures without AMP, three metal-binding sites are occupied by Zn(2+) and two of three metal sites (sites 1 and 2) by Mg(2+). Evidently, the association of AMP with FBPase disorders loop 52-72, the consequence of which is the release of cations from two of three metal binding sites. In the Mg(2+) complexes (but not the Zn(2+) complexes), the 1-OH group of fructose 6-phosphate (F6P) coordinates to the metal at site 1 and is oriented for a nucleophilic attack on the bound phosphate molecule. A mechanism is presented for the forward reaction, in which Asp74 and Glu98 together generate a hydroxide anion coordinated to the Mg(2+) at site 2, which then displaces F6P. Development of negative charge on the 1-oxygen of F6P is stabilized by its coordination to the Mg(2+) at site 1.     

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.     

D-Ribulose 5-phosphate 3-epimerase: functional and structural relationships to members of the ribulose-phosphate binding (beta/alpha)8-barrel superfamily

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.