[Show abstract][Hide abstract] ABSTRACT: PCNA ubiquitination in response to DNA damage leads to the recruitment of specialized translesion polymerases to the damage locus. This constitutes one of the initial steps in translesion synthesis (TLS)--a critical pathway for cell survival and for maintenance of genome stability. The recent crystal structure of ubiquitinated PCNA (Ub-PCNA) sheds light on the mode of association between the two proteins but also revealed that paradoxically, the ubiquitin surface engaged in PCNA interactions was the same as the surface implicated in translesion polymerase binding. This finding implied a degree of flexibility inherent in the Ub-PCNA complex that would allow it to transition into a conformation competent to bind the TLS polymerase. To address the issue of segmental flexibility, we combined multiscale computational modeling and small angle X-ray scattering. This combined strategy revealed alternative positions for ubiquitin to reside on the surface of the PCNA homotrimer, distinct from the position identified in the crystal structure. Two mutations originally identified in genetic screens and known to interfere with TLS are positioned directly beneath the bound ubiquitin in the alternative models. These computationally derived positions, in an ensemble with the crystallographic and flexible positions, provided the best fit to the solution scattering, indicating that ubiquitin dynamically associated with PCNA and is capable of transitioning between a few discrete sites on the PCNA surface. The finding of new docking sites and the positional equilibrium of PCNA-Ub occurring in solution provide unexpected insight into previously unexplained biological observations.
Proceedings of the National Academy of Sciences 10/2011; 108(43):17672-7. DOI:10.1073/pnas.1110480108 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Undecaprenyl pyrophosphate synthase is a cis-prenyltransferase enzyme, which is required for cell wall biosynthesis in bacteria. Undecaprenyl pyrophosphate synthase is an attractive target for antimicrobial therapy. We performed long molecular dynamics simulations and docking studies on undecaprenyl pyrophosphate synthase to investigate its dynamic behavior and the influence of protein flexibility on the design of undecaprenyl pyrophosphate synthase inhibitors. We also describe the first X-ray crystallographic structure of Escherichia coli apo-undecaprenyl pyrophosphate synthase. The molecular dynamics simulations indicate that undecaprenyl pyrophosphate synthase is a highly flexible protein, with mobile binding pockets in the active site. By carrying out docking studies with experimentally validated undecaprenyl pyrophosphate synthase inhibitors using high- and low-populated conformational states extracted from the molecular dynamics simulations, we show that structurally dissimilar compounds can bind preferentially to different and rarely sampled conformational states. By performing structural analyses on the newly obtained apo-undecaprenyl pyrophosphate synthase and other crystal structures previously published, we show that the changes observed during the molecular dynamics simulation are very similar to those seen in the crystal structures obtained in the presence or absence of ligands. We believe that this is the first time that a rare 'expanded pocket' state, key to drug design and verified by crystallography, has been extracted from a molecular dynamics simulation.
Chemical Biology & Drug Design 02/2011; 77(6):412-20. DOI:10.1111/j.1747-0285.2011.01101.x · 2.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Enabled by impressive advances in processing speed and low-latency intercomputer communications, computational biophysics has trended toward studies at longer and longer timescales. Although still relatively rare, the massive computing power now available to investigators has allowed for the brute force calculation of the 1012-molecular dynamics steps necessary to reach the millisecond-time regime (Klepeis et al., 2009). As this ability becomes more widespread, it promises to probe deeper into the nature and functional consequences of biomolecular motions, especially in conformational changes that have large kinetic barriers and occur relatively slowly. While long timescale investigations of large systems are clearly an exciting and powerful prospect, the field of biomolecular simulation is not limited to leveraging the horsepower of immense computational resources. Clever analyses using established techniques with well-chosen approximations have continually proven to be of great value in investigating interesting biophysical and biochemical problems. The article by Raimondi, Orozco, and Fanelli (2010) published in this issue of Structure is an excellent example of this mode of investigation. The authors use the familiar techniques of principal component analysis (PCA) and normal mode analysis (NMA) (Cui and Bahar, 2006) to identify the important structural flexibilities that enable proteins in the Ras superfamily to switch between their active and inactive states. In addition, this analysis leads to an interesting hypothesis regarding the evolutionary adaptation of structural deformations by the individual members of the superfamily to fulfill their specialized function.
[Show abstract][Hide abstract] ABSTRACT: Within influenza viral particles, the intricate balance between host cell binding and sialic acid receptor destruction is carefully maintained by the hemagglutinin (HA) and neuraminidase (NA) glycoproteins, respectively. A major outstanding question in influenza biology is the function of a secondary sialic acid binding site on the NA enzyme. Through a series of Brownian dynamics (BD) simulations of the avian N1, human pandemic N2, and currently circulating pandemic (H1)N1 enzymes, we have probed the role of this secondary sialic acid binding site in the avian N1 subtype. Our results suggest that electrostatic interactions at the secondary and primary sites in avian NA may play a key role in the recognition process of the sialic acid receptors and catalytic efficiency of NA. This secondary site appears to facilitate the formation of complexes with the NA protein and the sialic acid receptors, as well as provide HA activity to a lesser extent. Moreover, this site is able to steer inhibitor binding as well, albeit with reduced capacity in N1, and may have potential implications for drug resistance or optimal inhibitor design. Although the secondary sialic acid binding site has previously been shown to be nonconserved in swine NA strains, our investigations of the currently circulating pandemic H1N1 strain of swine origin appears to have retained some of the key features of the secondary sialic acid binding site. Our results indicate possible lowered HA activity for this secondary sialic acid site, which may be an important event in the emergence of the current pandemic strain.
Journal of the American Chemical Society 02/2010; 132(9):2883-5. DOI:10.1021/ja9073672 · 12.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Normal mode analysis offers an efficient way of modeling the conformational flexibility of protein structures. We use anisotropic displacement parameters from crystallography to test the quality of prediction of both the magnitude and directionality of conformational flexibility. Normal modes from four simple elastic network model potentials and from the CHARMM force field are calculated for a data set of 83 diverse, ultrahigh-resolution crystal structures. While all five potentials provide good predictions of the magnitude of flexibility, all-atom potentials have a clear edge at prediction of directionality, and the CHARMM potential has the highest prediction quality. The low-frequency modes from different potentials are similar, but those computed from the CHARMM potential show the greatest difference from the elastic network models. The comprehensive evaluation demonstrates the costs and benefits of using normal mode potentials of varying complexity.
[Show abstract][Hide abstract] ABSTRACT: With the increased popularity of normal mode analyses in structural biology, it is important to carefully consider how to best utilize the results for gaining biological insights without over interpretation. The discussion in this article argues that for the purpose of identifying correlated motions in biomolecules, a case separate from concomitant conformational changes of structural motifs, it is generally important to use a large number of normal modes. This is illustrated through three increasingly complex examples. The simplest case includes two bilinearly coupled harmonic oscillators and serves as a straightforward problem where the important considerations are explicit and transparent. The argument is then generalized to include a system of N-coupled harmonic oscillators and finally to a realistic biomolecule. Although a small number of normal modes are useful for probing structural flexibility, it is clear that a much larger number of modes are required for properly investigating correlated motions in biomolecules.
[Show abstract][Hide abstract] ABSTRACT: More than two decades of different types of mode analyses has shown that these techniques can be useful in describing large-scale motions in protein systems. A number of mode analyses are available and include quasiharmonics, classical normal mode, block normal mode, and the elastic network model. Each of these methods has been validated for protein systems and this variety allows researchers to choose the technique that gives the best compromise between computational cost and the level of detail in the calculation. These same techniques have not been systematically tested for nucleic acid systems, however. Given the differences in interactions and structural features between nucleic acid and protein systems, the validity of these techniques in the protein regime cannot be directly translated into validity in the nucleic acid realm. In this work, we investigate the usefulness of the above mode analyses as applied to two RNA systems, i.e., the hammerhead ribozyme and a guanine riboswitch. We show that classical normal-mode analysis can match the magnitude and direction of residue fluctuations from the more detailed, anharmonic technique, quasiharmonic analysis of a molecular dynamics trajectory. The block normal-mode approximation is shown to hold in the nucleic acid systems studied. Only the mode analysis at the lowest level of detail, the elastic network model, produced mixed results in our calculations. We present data that suggest that the elastic network model, with the popular parameterization, is not best suited for systems that do not have a close packed structure; this observation also hints at why the elastic network model has been found to be valid for many globular protein systems. The different behaviors of block normal-mode analysis and the elastic network model, which invoke similar degrees of coarse-graining to the dynamics but use different potentials, suggest the importance of applying a heterogeneous potential function in a robust analysis of the dynamics of biomolecules, especially those that are not closely packed. In addition to these comparisons, we briefly discuss insights into the conformational space available to the hammerhead ribozyme.
[Show abstract][Hide abstract] ABSTRACT: Paradoxically, glycine betaine (N,N,N-trimethyl glycine; GB) in vivo is both an effective osmoprotectant (efficient at increasing cytoplasmic osmolality and growth rate) and a compatible solute (without deleterious effects on biopolymer function, including stability and activity). For GB to be an effective osmoprotectant but not greatly affect biopolymer stability, we predict that it must interact very differently with folded protein surface than with that exposed in unfolding. To test this hypothesis, we quantify the preferential interaction of GB with the relatively uncharged surface exposed in unfolding the marginally stable lacI helix-turn-helix (HTH) DNA binding domain using circular dichroism and with the more highly charged surfaces of folded hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) using all-gravimetric vapor pressure osmometry (VPO) and compare these results with results of VPO studies (Hong et al. (2004), Biochemistry, 43, 14744-14758) of the interaction of GB with polyanionic duplex DNA. For these four biopolymer surfaces, we observe that the extent of exclusion of GB per unit of biopolymer surface area increases strongly with increasing fraction of anionic oxygen (protein carboxylate or DNA phosphate) surface. In addition, GB is somewhat more excluded from the surface exposed in unfolding the lacI HTH and from the folded surface of HEWL than expected from their small fraction of anionic surface, consistent with moderate exclusion of GB from polar amide surface, as predicted by the osmophobic model of protein stability (Bolen and Baskakov (2001) J. Mol. Biol. 310, 955-963). Strong exclusion of GB from anionic surface explains how it can be both an effective osmoprotectant and a compatible solute; analysis of this exclusion yields a lower bound on the hydration of anionic protein carboxylate surface of two layers of water (>or=0.22 H(2)O A(-)(2)).
[Show abstract][Hide abstract] ABSTRACT: To explore the domain-scale flexibility of bacterial RNA polymerase (RNAP) throughout its functional cycle, block normal-mode analyses (BNM) were performed on several important functional states, including the holoenzyme, the core complex, a model of RNAP bound to primarily duplex DNA, and a model of the ternary elongation complex. The calculations utilized a molecular mechanics (MM) force field with physical interactions; this is made possible by the use of BNM and the implementation of a sparse-matrix diagonalization routine. The use of homology models necessitated the MM force field rather than the simpler elastic network model (ENM). From the MM/BNM, we have systematically and semiquantitatively calculated the atomic fluctuations in the four functional states without bias due to crystal packing or other artifactual forces. We have observed that both alpha subunits and the omega subunit are rigid, in line with their roles as structural motifs that are not mechanistically involved in RNAP's functional cycle. It has been observed that the beta subunit has two highly mobile domains; these are commonly known as the beta1 and beta2 domains. Our calculations suggest that the flexibility of these domains is modulated throughout the functional cycle and that they move entirely independently of each other unless DNA is bound. From an energetic perspective, we have shown the beta2 domain can flex into and out of the cleft, forming interactions with DNA in the TEC as has been previously proposed. Our calculations also confirm that the beta' subunit's likely flexibility into and out of the DNA binding cleft is energetically allowed. These two observations validate that both of the RNAP crab claw's pincers are mobile, as both beta and beta' have substantial flexibility.