Mutagenesis studies of protein farnesyltransferase implicate aspartate beta 352 as a magnesium ligand

Protein farnesyltransferase (FTase) catalyzes the addition of a farnesyl chain onto the sulfur of a C-terminal cysteine of a protein substrate. Magnesium ions enhance farnesylation catalyzed by FTase by several hundred-fold, with a KMg value of 4 mM. The magnesium ion is proposed to coordinate the diphosphate leaving group of farnesyldiphosphate (FPP) to stabilize the developing charge in the farnesylation transition state. Here we further investigate the magnesium binding site using mutagenesis and biochemical studies. Free FPP binds Mg2+ with a Kd of 120 microM. The 10-fold weaker affinity for Mg2+ observed for the FTase.FPP.peptide ternary complex is probably caused by the positive charges in the diphosphate binding pocket of FTase. Furthermore, mutation of aspartate beta 352 to alanine (D beta 352A) or lysine (D beta 352K) in FTase drastically alters the Mg2+ dependence of FTase catalysis without dramatically affecting the rate constant of farnesylation minus magnesium or the binding affinity of either substrate. In D beta 352A FTase, the KMg increases 28-fold to 110 +/- 30 mM, and the farnesylation rate constant at saturating Mg2+ decreases 27-fold to 0.30 +/- 0.05 s-1. Substitution of a lysine for Asp-beta 352 removes the magnesium activation of farnesylation catalyzed by FTase but does not significantly enhance the rate constant for farnesylation in the absence of Mg2+. In wild type FTase, Mg2+ can be replaced by Mn2+ with a 2-fold lower KMn (2 mM). These results suggest both that Mg2+ coordinates the side chain carboxylate of Asp-beta 352 and that the role of magnesium in the reaction includes positioning the FPP prior to catalysis.     

Positively charged side chains in protein farnesyltransferase enhance catalysis by stabilizing the formation of the diphosphate leaving group

Protein farnesyltransferase (FTase) requires both Zn(2+) and Mg(2+) for efficient catalysis of the formation of a thioether bond between carbon-1 of farnesyldiphosphate (FPP) and the cysteine thiolate contained in the carboxy-terminal CaaX sequence of target proteins. Millimolar concentrations of Mg(2+) accelerate catalysis by as much as 700-fold in FTase. Although FTase lacks a typical DDXXD Mg(2+) binding site found in other enzymes that use Mg(2+) for diphosphate stabilization, D352beta in FTase has been implicated in binding Mg(2+) (Pickett et al. (2003) J. Biol. Chem. 278, 51243). Structural studies demonstrate that the diphosphate (PPi) group of FPP resides in a binding pocket made up of highly positively charged side chains, including residues R291beta and K294beta, prior to formation of an active conformation. Analysis of the Mg(2+) dependence of FTase mutants demonstrates that these positively charged residues decrease the Mg(2+) affinity up to 40-fold. In addition, these residues enhance the farnesylation rate constant by almost 80-fold in the presence of Mg(2+), indicating that these residues are not simply displaced by Mg(2+) during the reaction. Mutations at R291beta increase the pK(a) observed in the magnesium affinity, suggesting that this arginine stabilizes the deprotonated form of the PPi leaving group. Furthermore, binding and catalysis data using farnesylmonophosphate (FMP) as a substrate indicate that the side chains of R291beta and K294beta interact mainly with the beta-phosphate of FPP during the chemical reaction. These results allow refinement of the model of the Mg(2+) binding site and demonstrate that positive charge stabilizes the developing charge on the diphosphate leaving group.     

Computational studies of the farnesyltransferase ternary complex part I: substrate binding

Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyl diphosphate (FPP) to cysteine residues at or near the C-terminus of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in the extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step toward further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291beta, K164alpha, K294beta, and H248beta. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well-defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in a vacuum and solution. Our conclusion is further supported by theoretical J coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.     

Dual effects of familial Alzheimer's disease mutations (D7H,D7N,and H6R) on amyloid β peptide: Correlation dynamics and zinc binding

Although the N‐terminal region of Amyloid β (Aβ) peptides plays dual roles as metal‐coordinating sites and conformational modulator, few studies have been performed to explore the effects of mutations at this region on the overall conformational ensemble of Aβ and the binding propensity of metal ions. In this work, we focus on how three familial Alzheimer's disease mutations (D7H, D7N, and H6R) alter the structural characteristics and thermodynamic stabilities of Aβ42 using molecular dynamics simulations. We observe that each mutation displays increased β‐sheet structures in both N and C termini. In particular, both the N terminus and central hydrophobic region of D7H can form stable β‐hairpin structures with its C terminus. The conserved turn structure at Val²⁴–Lys²⁸ in all peptides and Zn²⁺‐bound Aβ42 is confirmed as the common structural motif to nucleate folding of Aβ. Each mutant can significantly increase the solvation free energy and thus enhance the aggregation of Aβ monomers. The correlation dynamics between Aβ(1–16) and Aβ(17–42) fragments are elucidated by linking the domain motions with the corresponding structured conformations. We characterize the different populations of correlated domain motions for each mutant from a more macroscopic perspective, and unexpectedly find that Zn²⁺‐bound Aβ42 ensemble shares the same populations as Aβ42, indicating that the binding of Zn²⁺ to Aβ follows the conformational selection mechanism, and thus is independent of domain motions, even though the structures of Aβ have been modified at a residue level. Proteins 2014; 82:3286–3297. © 2014 Wiley Periodicals, Inc.     

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

Computational studies of the farnesyltransferase ternary complex part II: the conformational activation of farnesyldiphosphate

Studies aimed at elucidating the reaction mechanism of farnesyltransferase (FTase), which catalyzes the prenylation of many cellular signaling proteins including Ras, has been an active area of research. Much is known regarding substrate binding and the impact of various catalytic site residues on catalysis. However, the molecular level details regarding the conformational rearrangement of farnesyldiphosphate (FPP), which has been proposed via structural analysis and mutagenesis studies to occur prior to the chemical step, is still poorly understood. Following on our previous computational characterization of the resting state of the FTase ternary complex, the thermodynamics of the conformational rearrangement step in the absence of magnesium was investigated for the wild type FTase and the Y300Fbeta mutant complexed with the peptide CVIM. In addition, we also explored the target dependence of the conformational activation step by perturbing isoleucine into a leucine (CVLM). The calculated free energy profiles of the proposed conformational transition confirm the presence of a stable intermediate state, which was identified only when the diphosphate is monoprotonated (FPP2-). The farnesyl group in the computed intermediate state assumes a conformation similar to that of the product complex, particularly for the first two isoprene units. We found that Y300beta can readily form hydrogen bonds with either of the phosphates of FPP. Removing the hydroxyl group on Y300beta does not significantly alter the thermodynamics of the conformational transition, but shifts the location of the intermediate farther away from the nucleophile by 0.5 A, which suggests that Y300beta facilitate the reaction by stabilizing the chemical step. Our results also showed an increased transition barrier height for CVLM (1.5 kcal/mol higher than that of CVIM). Although qualitatively consistent with the findings from the recent kinetic isotope experiments by Fierke and co-workers, the magnitude is not large enough to affect the rate-limiting step.     

A 1.3-A structure of zinc-bound N-terminal domain of calmodulin elucidates potential early ion-binding step

Calmodulin (CaM) is a 16.8-kDa calcium-binding protein involved in calcium-signal transduction. It is the canonical member of the EF-hand family of proteins, which are characterized by a helix-loop-helix calcium-binding motif. CaM is composed of N- and C-terminal globular domains (N-CaM and C-CaM), and within each domain there are two EF-hand motifs. Upon binding calcium, CaM undergoes a significant, global conformational change involving reorientation of the four helix bundles in each of its two domains. This conformational change upon ion binding is a key component of the signal transduction and regulatory roles of CaM, yet the precise nature of this transition is still unclear. Here, we present a 1.3-Å structure of zinc-bound N-terminal calmodulin (N-CaM) solved by single-wavelength anomalous diffraction phasing of a selenomethionyl N-CaM. Our zinc-bound N-CaM structure differs from previously reported CaM structures and resembles calcium-free apo-calmodulin (apo-CaM), despite the zinc binding to both EF-hand motifs. Structural comparison with calcium-free apo-CaM, calcium-loaded CaM, and a cross-linked calcium-loaded CaM suggests that our zinc-bound N-CaM reveals an intermediate step in the initiation of metal ion binding at the first EF-hand motif. Our data also suggest that metal ion coordination by two key residues in the first metal-binding site represents an initial step in the conformational transition induced by metal binding. This is followed by reordering of the N-terminal region of the helix exiting from this first binding loop. This conformational switch should be incorporated into models of either stepwise conformational transition or flexible, dynamic energetic state sampling-based transition.     

X-ray crystallographic studies of substrate binding to aristolochene synthase suggest a metal ion binding sequence for catalysis

The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D. The 1.89-A resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg(2+)(B)accompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-A resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PP(i), Mg(2+)(B), and Mg(2+)(C) to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent "snapshots" of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.     

Structural and mechanistic analysis of trichodiene synthase using site-directed mutagenesis: probing the catalytic function of tyrosine-295 and the asparagine-225/serine-229/glutamate-233-Mg2+B motif

Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the "aspartate-rich" motif D(100)DXX(D/E) that coordinates to Mg2+A and Mg2+C, and the "NSE/DTE" motif N(225)DXXSXXXE that chelates Mg2+B (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.     

Modulation in the conformational and stability attributes of the Alzheimer’s disease associated amyloid-beta mutants and their favorable stabilization by curcumin: molecular dynamics simulation analysis

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive accumulation of amyloid-beta (Aβ) peptides in brain. In the present study, two familial Aβ42 mutations, namely A2V (harmful) and A2T (protective) have been analyzed and compared with the wild-type (WT) by performing all-atom molecular dynamics (MD) simulations in the absence and presence of curcumin, a well-known inhibitor of Aβ plaque formation. Mutant A2V was found to exhibit highest stability followed by WT and mutant A2T in the absence of curcumin. This stability trend was found to be reversed in the presence of curcumin, suggesting a significant change in the conformational landscape of Aβ42 folding. Due to significant differences in the folding and interaction patterns of the mutants A2V and A2T, curcumin exhibited higher binding affinity for mutant A2T as compared to that of A2V. To the best of our knowledge, this is the first report on the effect of curcumin binding on structural landscapes of the two contrasting point mutants providing an understanding of the basis of Aβ plaque formation and its prevention by curcumin.     

Kinetic studies of protein farnesyltransferase mutants establish active substrate conformation

The zinc metalloenzyme protein farnesyltransferase (FTase) catalyzes the transfer of a 15-carbon farnesyl moiety from farnesyl diphosphate (FPP) to a cysteine residue near the C-terminus of a protein substrate. Several crystal structures of inactive FTase.FPP.peptide complexes indicate that K164alpha interacts with the alpha-phosphate and that H248beta and Y300beta form hydrogen bonds with the beta-phosphate of FPP [Strickland, C. L., et al. (1998) Biochemistry 37, 16601-16611]. Mutations K164Aalpha, H248Abeta, and Y300Fbeta were prepared and analyzed by single turnover kinetics and ligand binding studies. These mutations do not significantly affect the enzyme affinity for FPP but do decrease the farnesylation rate constant by 30-, 10-, and 500-fold, respectively. These mutations have little effect on the pH and magnesium dependence of the farnesylation rate constant, demonstrating that the side chains of K164alpha, Y300beta, and H248beta do not function either as general acid-base catalysts or as magnesium ligands. Mutation of H248beta and Y300beta, but not K164alpha, decreases the farnesylation rate constant using farnesyl monophosphate (FMP). These data suggest that, contrary to the conclusions derived from analysis of the static crystal structures, the transition state for farnesylation is stabilized by interactions between the alpha-phosphate of the isoprenoid substrate and the side chains of Y300beta and H248beta. These results suggest an active substrate conformation for FTase wherein the C1 carbon of the FPP substrate moves toward the zinc-bound thiolate of the protein substrate to react, resulting in a rearrangement of the diphosphate group relative to its ground state position in the binding pocket.     

A novel catalytic mechanism for ATP hydrolysis employed by the N-terminal nucleotide-binding domain of Cdr1p, a multidrug ABC transporter of Candida albicans

Although essentially conserved, the N-terminal nucleotide-binding domain (NBD) of Cdr1p and other fungal transporters has some unique substitutions of amino acids which appear to have functional significance for the drug transporters. We have previously shown that the typical Cys193 in Walker A as well as Trp326 and Asp327 in the Walker B of N-terminal NBD (NBD-512) of Cdr1p has acquired unique roles in ATP binding and hydrolysis. In the present study, we show that due to spatial proximity, fluorescence resonance energy transfer (FRET) takes place between Trp326 of Walker B and MIANS [2-(4-maleimidoanilino) naphthalene-6-sulfonic acid] on Cys193 of Walker A motif. By exploiting FRET, we demonstrate how these critical amino acids are positioned within the nucleotide-binding pocket of NBD-512 to bind and hydrolyze ATP. Our results show that both Mg2+ coordination and nucleotide binding contribute to the formation of the active site. The entry of Mg2+ into the active site causes the first large conformational change that brings Trp326 and Cys193 in close proximity to each other. We also show that besides Trp326, typical Glu238 in the Q-loop also participates in coordination of Mg2+ by NBD-512. A second conformational change is induced when ATP, but not ADP, docks into the pocket. Asn328 does sensing of the gamma-phosphate of the substrate in the extended Walker B motif, which is essential for the second conformational change that must necessarily precede ATP hydrolysis. Taken together our results imply that the uniquely placed residues in NBD-512 have acquired critical roles in ATP catalysis, which drives drug extrusion.     

Expression of Na+,K+-ATPase in Pichia pastoris: analysis of wild type and D369N mutant proteins by Fe2+-catalyzed oxidative cleavage and molecular modeling

Na+,K+-ATPase (pig alpha1,beta1) has been expressed in the methylotrophic yeast Pichia pastoris. A protease-deficient strain was used, recombinant clones were screened for multicopy genomic integrants, and protein expression, and time and temperature of methanol induction were optimized. A 3-liter culture provides 300-500 mg of membrane protein with ouabain binding capacity of 30-50 pmol mg-1. Turnover numbers of recombinant and renal Na+,K+-ATPase are similar, as are specific chymotryptic cleavages. Wild type (WT) and a D369N mutant have been analyzed by Fe2+- and ATP-Fe2+-catalyzed oxidative cleavage, described for renal Na+,K+-ATPase. Cleavage of the D369N mutant provides strong evidence for two Fe2+ sites: site 1 composed of residues in P and A cytoplasmic domains, and site 2 near trans-membrane segments M3/M1. The D369N mutation suppresses cleavages at site 1, which appears to be a normal Mg2+ site in E2 conformations. The results suggest a possible role of the charge of Asp369 on the E1 <--> E2 conformational equilibrium. 5'-Adenylyl-beta,gamma-imidodi-phosphate(AMP-PNP)-Fe2+-catalyzed cleavage of the D369N mutant produces fragments in P (712VNDS) and N (near 440VAGDA) domains, described for WT, but only at high AMP-PNP-Fe2+ concentrations, and a new fragment in the P domain (near 367CSDKTGT) resulting from cleavage. Thus, the mutation distorts the active site. A molecular dynamic simulation of ATP-Mg2+ binding to WT and D351N structures of Ca2+-ATPase (analogous to Asp369 of Na+,K+-ATPase) supplies possible explanations for the new cleavage and for a high ATP affinity, which was observed previously for the mutant. The Asn351 structure with bound ATP-Mg2+ may resemble the transition state of the WT poised for phosphorylation.     

Nucleotide binding to the G12V-mutant of Cdc42 investigated by X-ray diffraction and fluorescence spectroscopy: two different nucleotide states in one crystal

The 2.5 A crystal structure of the full length human placental isoform of the Gly12 to Val mutant Cdc42 protein (Cdc42(G12V)) bound to both GDP/Mg2+ and GDPNH2 (guanosine-5'-diphospho-beta-amidate) is reported. The crystal contains two molecules in the asymmetric unit, of which one has bound GDP/Mg2+, while the other has bound GDPNH2 without a Mg2+ ion. Crystallization of the protein was induced via hydrolysis of the Cdc42 x GppNHp complex by the presence of contaminating alkaline phosphatase activity in combination with the crystallization conditions. This prompted us to compare the binding characteristics of GDPNH2 vs. GDP. The amino group of GDPNH2 drastically reduces the affinity to Cdc42 in comparison with that of GDP, causes the loss of the Mg2+ ion, and apparently also increases the conformational flexibility of the protein as seen in the crystal. Both the switch I and switch II regions are visible in the electron density of the GDP-bound molecule, but not in the molecule bound to GDPNH2. The C-terminus containing the CaaX-motif is partly ordered in both molecules due to an intramolecular disulfide bond formed between Cys105/Cys188 and Cys305/Cys388, respectively.     

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.     

Molecular dynamics simulations of wild type and mutant of Pin1 peptidyl-prolyl isomerase

Pin1 catalyses the intrinsically slow process of cis-trans isomerisation and has been identified as a possible drug target in many diseases. Recently, the wild type (WT) and the Cys113Asp mutant of the Pin1 peptidyl-prolyl isomerase (PPIase) domain were determined by nuclear magnetic resonance. In this article, the WT and Cys113Asp mutant of PPIase domain are studied by molecular dynamics simulations. The structural stability analysis shows that the Cys113Asp mutation leads to the higher fluctuation of hydrophobic core in PPIase domain. The intrinsic correlated motions are important for the catalytic function of Pin1, whereas the Cys113Asp mutant system loses pivotal dynamical properties and develops wider conformational states than those in WT system. The intramolecular hydrogen bonds play crucial roles in the structural stability of PPIase domain. The mutated residue Asp113 attracts the side chain of His59 in the Cys113Asp system, which unbalances the internal interactions inside the catalytic tetrad. Meanwhile, the conformational changes of PPIase domain affect the side chain orientations of Lys63 and Arg69, which limit their binding with substrates. The Cys113Asp mutation destabilises the whole binding region of Pin1 PPIase domain, so the catalysis activity is severely reduced. These results are consistent with experimental studies and may help to understand the isomerisation mechanisms of Pin1.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

The kinetics of cytochalasin D binding to monomeric actin

It has been shown previously, using G-actin labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylene-diamine, that Mg2+ induces a conformational change in monomeric G-actin as a consequence of binding to a tight divalent cation binding site (Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886). Using the same fluorescent probe, we show that, subsequent to the Mg2+-induced conformational change, cytochalasin D induces a fluorescence decrease. The data are consistent with a mechanism which proposes that, after Mg2+ binding, cytochalasin D binds and induces a second conformational change which results in overall tight binding of the cytochalasin. The initial binding of cytochalasin D to monomeric actin labeled with the fluorescent probe was found to be 200 microM, and the forward and reverse rate constants for the subsequent conformational change were 350 s-1 and 8 s-1, respectively, with an overall dissociation constant to the Mg2+-induced form of 4.6 microM. The conformational change does not occur in monomeric actin in the presence of Ca2+ rather than Mg2+, but Ca2+ competes with Mg2+ for the tight binding site on the G-actin molecule. Direct binding studies show that actin which has not been labeled with the fluorophore binds cytochalasin D more tightly. The conformational change induced by Mg2+ and cytochalasin D precedes the formation of an actin dimer.     

Molecular dynamics simulations on the critical states of the farnesyltransferase enzyme

Protein farnesyltransferase (FTase) is a particularly interesting zinc enzyme that promotes the transfer of a 15-carbons isoprenoid farnesyl group from farnesyl diphosphate (FPP) to a number of peptide substrates with a typical-CAAX motif at the carboxyl-terminus, where C represents the cysteine residue that is farnesylated. This enzyme has been the subject of great attention in anticancer research, as several proteins known to be involved in human cancer development are thought to serve as substrates for FTase and to require farnesylation for proper biological activity. Several FTase inhibitors have advanced into clinical testing. However, despite the progress in the field several functional and mechanistic doubts on the FTase catalytic activity have persisted. This work describes the application of molecular dynamics simulations using specifically designed molecular mechanical parameters to the four key-intermediate states formed during the FTase catalytic mechanism–FTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTase-Product). The study involves a comparative analysis of several important molecular aspects for which are vital not only motion but also the conformational sampling of both enzyme and substrate as well as their interaction, and especially the effect of the solvent. These include the radial distribution function of the water molecules around the catalytically important zinc metal atom, the conformations of the substrate and product molecules and the corresponding RMSF values, critical hydrogen bonds and several catalytically relevant distances. These results are discussed in light of recent experimental and computational evidence, yielding new insights into the elusive catalytic mechanism of this enzyme.     

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.