ATP binding enables broad antibiotic selectivity of aminoglycoside phosphotransferase(3')-IIIa: an elastic network analysis

The bacterial enzyme aminoglycoside phosphotransferase(3′)-IIIa (APH) confers resistance against a wide range of aminoglycoside antibiotics. In this study, we use the Gaussian network model to investigate how the binding of nucleotides and antibiotics influences the dynamics and thereby the ligand binding properties of APH. Interestingly, in NMR experiments, the dynamics differ significantly in various APH complexes, although crystallographic studies indicate that no larger conformational changes occur upon ligand binding. Isothermal titration calorimetry also shows different thermodynamic contributions to ligand binding. Formation of aminoglycoside-APH complexes is enthalpically driven, while the enthalpic change upon aminoglycoside binding to the nucleotide-APH complex is much smaller. The differential effects of nucleotide binding and antibiotic binding to APH can be explained theoretically by single-residue fluctuations and correlated motions of the enzyme. The surprising destabilization of β-sheet residues upon nucleotide binding, as seen in hydrogen/deuterium exchange experiments, shows that the number of closest neighbors does not fully explain residue flexibility. Additionally, we must consider correlated motions of dynamic protein domains, which show that not only connectivity but also the overall protein architecture is important for protein dynamics.     

Using molecular dynamics to map interaction networks in an aminoacyl-tRNA synthetase

Long-range functional communication is a hallmark of many enzymes that display allostery, or action-at-a-distance. Many aminoacyl-tRNA synthetases can be considered allosteric, in that their trinucleotide anticodons bind the enzyme at a site removed from their catalytic domains. Such is the case with E. coli methionyl-tRNA synthase (MetRS), which recognizes its cognate anticodon using a conserved tryptophan residue 50 A away from the site of tRNA aminoacylation. The lack of details regarding how MetRS and tRNA(Met) interact has limited efforts to deconvolute the long-range communication that occurs in this system. We have used molecular dynamics simulations to evaluate the mobility of wild-type MetRS and a Trp-461 variant shown previously by experiment to be deficient in tRNA aminoacylation. The simulations reveal that MetRS has significant mobility, particularly at structural motifs known to be involved in catalysis. Correlated motions are observed between residues in distant structural motifs, including the active site, zinc binding motif, and anticodon binding domain. Both mobility and correlated motions decrease significantly but not uniformly upon substitution at Trp-461. Mobility of some residues is essentially abolished upon removal of Trp-461, despite being tens of Angstroms away from the site of mutation and solvent exposed. This conserved residue does not simply participate in anticodon binding, as demonstrated experimentally, but appears to mediate the protein's distribution of structural ensembles. Finally, simulations of MetRS indicate that the ligand-free protein samples conformations similar to those observed in crystal structures with substrates and substrate analogs bound. Thus, there are low energetic barriers for MetRS to achieve the substrate-bound conformations previously determined by structural methods.     

Analysis of the code relating sequence to conformation in globular proteins. An informational analysis of the role of the residue in determining the conformation of its neighbours in the primary sequence

1. The effect exerted by a residue on the conformation of neighbouring residues was analysed by using data from nine globular proteins of known sequence and conformation. 2. An information measure was used which estimated the role of a residue in influencing neighbouring conformations and also its tendency to influence the lengths of runs of residues in that conformation. This measure was estimated for each residue in all conformations defined by domains on the varphi, psi diagram. 3. Plots of the information measure yielded an intercept, which was a measure of intra-residue information for a residue. The slope was a measure of the statistical co-operativity or tendency of the residue to influence the occurrence of its neighbours in runs of a particular conformation. Both parameters are a function of the residue type. Statistical co-operativity is found in the alpha(1)-helical (H(1)) and beta-pleated-sheet (P(2)) conformations and, to a lesser extent, in their distorted variants H(2) and P(1). 4. The directional nature of these influences for H(1) and P(2) conformations is illustrated by plots of the information measure against the distance m from the residue, for m=-10 to +10. 5. The results for statistical co-operativity are discussed in relation to theories of helix-coil and pleated-sheet-coil transitions. The value of the information-theory-derived parameters in obtaining s parameters for the Zimm & Bragg (1959) equations is illustrated. 6. Directional effects are discussed with particular relation to mechanisms of the termination of helices and the involvement of the alpha(II) conformation and also to discontinuities in pleated-sheet conformations.     

Substrate-dependent millisecond domain motions in DNA polymerase β

DNA polymerase β (Pol β) is a 39-kDa enzyme that performs the vital cellular function of repairing damaged DNA. Mutations in Pol β have been linked to various cancers, and these mutations are further correlated with altered Pol β enzymatic activity. The fidelity of correct nucleotide incorporation into damaged DNA is essential for Pol β repair function, and several studies have implicated conformational changes in Pol β as a determinant of this repair fidelity. In this work, the rate constants for domain motions in Pol β have been determined by solution NMR relaxation dispersion for the apo and substrate-bound, binary forms of Pol β. In apo Pol β, molecular motions, primarily isolated to the DNA lyase domain, are observed to occur at 1400 s(-1). Additional analysis suggests that these motions allow apo Pol β to sample a conformation similar to the gapped DNA-substrate-bound form. Upon binding DNA, these lyase domain motions are significantly quenched, whereas evidence for conformational motions in the polymerase domain becomes apparent. These NMR studies suggest an alteration in the dynamic landscape of Pol β due to substrate binding. Moreover, a number of the flexible residues identified in this work are also the location of residues, which upon mutation lead to cancer phenotypes in vivo, which may be due to the intimate role of protein motions in Pol β fidelity.     

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.     

Interaction of tyrosine 65 of RecA protein with the first and second DNA strands

We investigated the structure of the active RecA-DNA complex by analyzing the environment of tyrosine residue 65, which is on the DNA-binding surface of the protein. We prepared a modified RecA protein in which the tyrosine residue was replaced by tryptophan, a natural fluorescent reporter, and measured the change in its fluorescence upon binding of DNA and cofactor. The fluorescence of the inserted tryptophan 65 (Trp65) was centered at 345 nm, indicating a partly exposed residue. Binding cofactor, adenosine 5'-O-3-thiotriphosphate (ATPgammaS), alone at a low salt concentration did not change the fluorescence of Trp65, confirming that the residue is not close to the nucleotide. In contrast, the binding of single-stranded DNA quenched the fluorescence of Trp65 in both the presence and absence of ATPgammaS. Trp65 fluorescence was also quenched upon binding a second DNA strand. The fluorescence change depended upon the presence and absence of ATPgammaS, reflecting the difference in the DNA binding. These results indicate that residue 65 is close to both the first and second DNA strands. The degree of quenching depended upon the base composition of DNA, suggesting that the residue 65 interacts with the DNA bases. Binding of DNA with ATPgammaS as well as binding of ATPgammaS alone at high salt concentration shifted the fluorescence emission peak from 345 to 330 nm, indicating a change from a polar to a non-polar environment. Therefore, the environment change around residue 65 would also be linked to a change in conformation and thus the activation of the protein.     

Protein dynamics and monomer-monomer interactions in AntR activation by electron paramagnetic resonance and double electron-electron resonance

The Anthracis repressor (AntR) is a Mn(II)-activated DNA binding protein that is involved in the regulation of Mn(II) homeostasis in Bacillus anthracis. AntR is structurally and functionally homologous to Mn(II)-activated repressor from Bacillus subtillis (MntR). Our studies on AntR focus on metal-regulated activation of the protein. Line shape analysis of continuous wave electron paramagnetic resonance (EPR) spectra showed that metal binding resulted in a general reduction of backbone dynamics and that there were no further changes in backbone motion upon DNA binding. Double electron-electron resonance (DEER) pulsed EPR spectroscopy was used to measure distances between nitroxide spin labels strategically placed in dimeric AntR. The DEER data were analyzed assuming Gaussian distributions for discrete populations of spins. A structural model for AntR was built from homology to MntR, and the experimentally measured distances were simulated to distinguish between spin label and backbone motions. Together with the computational analysis, the DEER results for apo-AntR indicated relatively narrow conformational distributions for backbone residues at the dimer interface and near the metal binding site. No significant changes were observed on these sites in the presence of metal or DNA. On the other hand, the distribution of the conformers and the distances between the putative DNA binding helices decreased upon metal binding. These results suggest that the DNA binding region of AntR shows large amplitude backbone motions in the absence of metal, which may preclude sequence-specific binding to promoter sites. Metal binding narrows the range of conformations accessible in this region and shortens the mean distance between the DNA binding helices, probably resulting in alignment that optimizes promoter recognition and binding.     

Influence of inhibitor binding on the internal motions of lysozyme

Time-resolved laser-induced fluorescence depolarization measurements of internal motions in lysozyme are presented. The fluorescent dye eosin binds in a one-to-one complex with the enzyme, and is used both to measure the overall tumbling time constants and to probe the motions of residues in the region of binding. The precision and accuracy of the present method for determining the overall tumbling time constants compare favorably with those from other methods used in the literature. The extent of the internal motions, as described by a model independent order parameter, S2, is temperature dependent, and changes when the inhibitor N,N',N"-triacetylchitotriose, (GlcNAc)3, is bound to the active site of the enzyme. The observed temperature dependence and changes in S2 upon binding of (GlcNAc)3 are interpreted in terms of a nonharmonic model of the effective potential that is consistent with the picture of concerted motions in the protein. The values of the parameters of the potential that reproduce the data with and without the bound inhibitor imply that (GlcNAc)3 binding causes an increase in the rigidity of the protein, which agree qualitatively with other results on the lysozyme-(GlcNAc)3 system.     

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

Abstract

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1?LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.     

Roles of the human Rad51 L1 and L2 loops in DNA binding

The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.     

All-atom simulations of structures and energetics of c-di-GMP-bound and free PleD

Cyclic diguanosine monophosphate is a bacterial second messenger involved in a lifestyle switch from single cells to biofilm formation. Atomistic simulations are used to characterize inhibited diguanylate cyclase (DGC) PleD with emphasis on the feedback inhibition mechanism. Normal-mode calculations show a rigidification particularly in both the inhibition site and the active site of the protein upon ligand binding. Extensive molecular dynamics simulations in explicit solvent and analysis of the dynamical cross-correlation maps suggest two distinct coupling pathways between the active and the inhibition site: direct information transfer either through the β-strands β2 and β3 of the DGC domain (pathway I) or via the disordered regions connecting domains D2 and DGC (pathway II). In addition, dynamical cross-correlation maps show differences in the correlation between neighboring domains upon ligand binding and upon the point mutation R390A. The correlated motions between domains D1 and D2, which form the dimerization interface, are stronger for free PleD. Complementary to the experimentally observed short-range interactions in ligated PleD, the present work also characterizes the long-range, delocalized interactions between domains that are important for understanding activation and allosteric control of the protein. Based on the results, experimental characterization of the point mutant R353 and of the double mutant N357/H394 is proposed to differentiate between pathways I and II.     

A K52Q substitution in the globular domain of histone H1t modulates its nucleosome binding properties

A comparison of the globular domain sequences of the somatic H1d and testis-specific H1t revealed a single substitution of lysine 52 in H1d to glutamine 54 in H1t, which is one of the three crucial residues within the second DNA binding site. The globular domains of both histones were modeled using the crystal structure of chicken GH5 as a template and was also docked onto the nucleosome structure. The glutamine residue in histone H1t forms a hydrogen bond with main chain carbonyl of methionine-52 (in H1t) and is spatially oriented away from the nucleosome dyad axis. A consequence of this change was a lower affinity of recombinant histone H1t towards Four-way junction DNA and reconstituted 5S mononucleosomes. When Gln-54 in Histone H1t was mutated to lysine, its binding affinity towards DNA substrates was comparable to that of histone H1d. The differential binding of histones H1d and H1t towards reconstituted mononucleosomes was also reflected in the chromatosome-stop assay.