Modifications of the amide bond and conformational constraints in pseudopeptide analogues

We have investigated the conformational effects of modifying the amide group in model dipeptides. The N-methyl amide ψ[CO-NMe], N-hydroxy amide ψ[CO-N(OH)], N-amino amide ψ[ CO-N (NH₂)], retro amide ψ[ NH-CO], reduced amide in the neutral ψ[CH₂-NH] and protonated ψ[CH₂-N ⁺ H₂] state, and hydrazide ψ[CO-NH-NH] have been introduced as surrogates of the amide link in pseudopeptide derivatives of the Pro-Gly or Ala-Gly model dipeptides protected on both termini by an amide group. These compounds have been studied in solution by proton nmr and ir spectroscopy, and in the solid state by x-ray diffraction, giving an extended data set of experimental structural and conformational information on pseudopeptide sequences. The conformational effects depend both on the nature and the position of the modified amide link. Some modifications appear to have no intrinsic conformational induction (N-amino and retro amide), but destabilize any local folded structure by hydrogen-bond breaking. Because of the formation of strong intramolecular interactions, others are capable of stabilizing a β-turn (for example protonated reduced amide), or of inducing a particular local conformation such as a β- or γ-like turn (for example N-hydroxy amide). The particular geometry of the cis N-methyl amide and of the “hydrazino” proline favors the formation of a sharp turn of the main chain. All these structural data are of interest to the design of bioactive peptide mimics. © 1993 John Wiley & Sons, Inc.     

Snapshots of a Y-family DNA polymerase in replication: substrate-induced conformational transitions and implications for fidelity of Dpo4

Y-family DNA polymerases catalyze translesion DNA synthesis over damaged DNA. Each Y-family polymerase has a polymerase core consisting of a palm, finger and thumb domain in addition to a fourth domain known as a little finger domain. It is unclear how each domain moves during nucleotide incorporation and what type of conformational changes corresponds to the rate-limiting step previously reported in kinetic studies. Here, we present three crystal structures of the prototype Y-family polymerase: apo-Dpo4 at 1.9 Å resolution, Dpo4-DNA binary complex and Dpo4-DNA-dTMP ternary complex at 2.2 Å resolution. Dpo4 undergoes dramatic conformational changes from the apo to the binary structures with a 131° rotation of the little finger domain relative to the polymerase core upon DNA binding. This DNA-induced conformational change is verified in solution by our tryptophan fluorescence studies. In contrast, the polymerase core retains the same conformation in all three conformationally distinct states. Particularly, the finger domain which is responsible for checking base pairing between the template base and an incoming nucleotide retains a rigid conformation. The inflexibility of the polymerase core likely contributes to the low fidelity of Dpo4, in addition to its loose and solvent-accessible active site. Interestingly, while the binary and ternary complexes of Dpo4 retain an identical global conformation, the aromatic side chains of two conserved tyrosines at the nucleotide-binding site change orientations between the binary and ternary structures. Such local conformational changes may correspond to the rate-limiting step in the mechanism of nucleotide incorporation. Together, the global and local conformational transitions observed in our study provide a structural basis for the distinct kinetic steps of a catalytic cycle of DNA polymerization performed by a Y-family polymerase.     

Structural role for a conserved region in the CTP synthetase glutamine amide transfer domain.

Site-directed mutations were introduced into a conserved region of the Escherichia coli CTP synthetase glutamine amide transfer domain. The amino acid replacements, valine 349 to serine, glycine 351 to alanine, glycine 352 to proline, and glycine 352 to cysteine, all increased the lability of CTP synthetase. The proline 352 replacement abolished the capacity to form the covalent glutaminyl-cysteine 379 catalytic intermediate, thus preventing glutamine amide transfer function; NH3-dependent CTP synthetase activity was retained. In CTP synthetase (serine 349), both glutamine and NH3-dependent activities were increased approximately 30% relative to that of the wild type. CTP synthetase mutants alanine 351 and cysteine 352 were not overproduced because of apparent instability and proteolytic degradation. We conclude that the conserved region between residues 346 and 355 in the CTP synthetase glutamine amide transfer domain has an important structural role.     

Structural basis for a functional antagonist in the transforming growth factor beta superfamily

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.     

Fluorescence of native single-Trp mutants in the lactose permease from Escherichia coli: structural properties and evidence for a substrate-induced conformational change.

Six single-Trp mutants were engineered by individually reintroducing each of the native Trp residues into a functional lactose permease mutant devoid of Trp (Trp-less permease; Menezes ME, Roepe PD, Kaback HR, 1990, Proc Natl Acad Sci USA 87:1638-1642), and fluorescent properties were studied with respect to solvent accessibility, as well as alterations produced by ligand binding. The emission of Trp 33, Trp 78, Trp 171, and Trp 233 is strongly quenched by both acrylamide and iodide, whereas Trp 151 and Trp 10 display a decrease in fluorescence in the presence of acrylamide only and no quenching by iodide. Of the six single-Trp mutants, only Trp 33 exhibits a significant change in fluorescence (ca. 30% enhancement) in the presence of the substrate analog beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). This effect was further characterized by site-directed fluorescent studies with purified single-Cys W33-->C permease labeled with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS). Titration of the change in the fluorescence spectrum reveals a 30% enhancement accompanied with a 5-nm blue shift in the emission maximum, and single exponential behavior with an apparent KD of 71 microM. The effect of substrate binding on the rate of MIANS labeling of single-Cys 33 permease was measured in addition to iodide and acrylamide quenching of the MIANS-labeled protein. Complete blockade of labeling is observed in the presence of TDG, as well as a 30% decrease in accessibility to iodide with no change in acrylamide quenching. Overall, the findings are consistent with the proposal (Wu J, Frillingos S, Kaback HR, 1995a, Biochemistry 34:8257-8263) that ligand binding induces a conformational change at the C-terminus of helix I such that Pro 28 and Pro 31, which are on one face, become more accessible to solvent, whereas Trp 33, which is on the opposite face, becomes less accessible to the aqueous phase. The findings regarding accessibility to collisional quenchers are also consistent with the predicted topology of the six native Trp residues in the permease.     

10-Formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates

The 10-formyltetrahydrofolate synthetase domain of the trifunctional enzyme C1-tetrahydrofolate synthase appears to undergo a conformational change in the presence of tetrahydropteroylpolyglutamates, MgATP, and ammonium ion. The binding of these ligands increases the denaturation temperature of the enzyme by 12 degrees C, abolishes the cold lability of the enzyme, and alters its susceptibility to digestion by chymotrypsin. The results suggest that a conformational change is dependent upon binding of the third glutamate residue of tetrahydropteroylpolyglutamates and the beta-phosphoryl group of MgATP. The Km values for MgATP and formate are lowered 3.6- and 520-fold, respectively, when tetrahydropteroyltriglutamate is used as the substrate in place of tetrahydropteroylmonoglutamate. A sensitive coupled assay involving C1-tetrahydrofolate synthase and serine hydroxymethyltransferase was developed to determine the activity of 10-formyltetrahydrofolate synthetase. The assay gives linear rates with the tetrahydropteroylpolyglutamates as substrates but not with the monoglutamate form.     

The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.     

Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.     

A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.     

Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism.

We have solved crystal structures of two complexes with Escherichia coli thymidylate synthase (TS) bound either to the cofactor analog N10-propargyl-5,8-dideazafolate (CB3717) or to a tighter binding polygutamyl derivative of CB3717. These structures suggest that cofactor binding alone is sufficient to induce the conformational change in TS; dUMP binding is not required. Because polyglutamyl folates are the primary cofactor form in vivo, and because they can bind more tightly than dUMP to TS, these structures may represent a key intermediate along the TS reaction pathway. These structures further suggest that the dUMP binding site is accessible in the TS-cofactor analog binary complexes. Conformational flexibility of the binary complex may permit dUMP to enter the active site of TS while the cofactor is bound. Alternatively, dUMP may enter the active site from the opposite side that the cofactor appears to enter; that is, through a portal flanked by arginines that also coordinate the phosphate group in the active site. Entry of dUMP through this portal may allow dUMP to bind to a TS-cofactor binary complex in which the complex has completed its conformational transition to the catalytically competent structure.     

Structural biology of Rad50 ATPase: ATP-driven conformational control in DNA double-strand break repair and the ABC-ATPase superfamily

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.     

Dissection of the stepwise mechanism to beta-lactam formation and elucidation of a rate-determining conformational change in beta-lactam synthetase

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.     

Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange

A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.     

Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin

Coordination between the nucleotide-binding site and the converter domain of myosin is essential for its ATP-dependent motor activities. To unveil the communication pathway between these two sites, we investigated contact between side chains of Phe-482 in the relay helix and Gly-680 in the SH1-SH2 helix. F482A myosin, in which Phe-482 was changed to alanine with a smaller side chain, was not functional in vivo. In vitro, F482A myosin did not move actin filaments and the Mg2+-ATPase activity of F482A myosin was hardly activated by actin. Phosphate burst and tryptophan fluorescence analyses, as well as fluorescence resonance energy transfer measurements to estimate the movements of the lever arm domain, indicated that the transition from the open state to the closed state, which precedes ATP hydrolysis, is very slow. In contrast, F482A/G680F doubly mutated myosin was functional in vivo and in vitro. The fact that a larger side chain at the 680th position suppresses the defects of F482A myosin suggests that the defects are caused by insufficient contact between side chains of Ala-482 and Gly-680. Thus, the contact between these two side chains appears to play an important role in the coordinated conformational changes and subsequent ATP hydrolysis.     

Cardiolipin binding in bacterial respiratory complexes: Structural and functional implications

The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).