Oleg V. Tsodikov

Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature.

New computer programs, SurfRace and FastSurf, perform fast calculations of the solvent accessible and molecular (solvent excluded) surface areas of macromolecules. Program SurfRace also calculates the areas of cavities inaccessible from the outside. We introduce the definition of average curvature of molecular surface and calculate average molecular surface curvatures for each atom in a structure. All surface area and curvature calculations are analytic and therefore yield exact values of these quantities. High calculation speed of this software is achieved primarily by avoiding computationally expensive mathematical procedures wherever possible and by efficient handling of surface data structures. The programs are written initially in the language C for PCs running Windows 2000/98/NT, but their code is portable to other platforms with only minor changes in input‐output procedures. The algorithm is robust and does not ignore either multiplicity or degeneracy of atomic overlaps. Fast, memory‐efficient and robust execution make this software attractive for applications both in computationally expensive energy minimization algorithms, such as docking or molecular dynamics simulations, and in stand‐alone surface area and curvature calculations.

Structural basis for the recruitment of ERCC1-XPF to nucleotide excision repair complexes by XPA

The nucleotide excision repair (NER) pathway corrects DNA damage caused by sunlight, environmental mutagens and certain antitumor agents. This multistep DNA repair reaction operates by the sequential assembly of protein factors at sites of DNA damage. The efficient recognition of DNA damage and its repair are orchestrated by specific protein–protein and protein–DNA interactions within NER complexes. We have investigated an essential protein–protein interaction of the NER pathway, the binding of the XPA protein to the ERCC1 subunit of the repair endonuclease ERCC1‐XPF. The structure of ERCC1 in complex with an XPA peptide shows that only a small region of XPA interacts with ERCC1 to form a stable complex exhibiting submicromolar binding affinity. However, this XPA peptide is a potent inhibitor of NER activity in a cell‐free assay, blocking the excision of a cisplatin adduct from DNA. The structure of the peptide inhibitor bound to its target site reveals a binding interface that is amenable to the development of small molecule peptidomimetics that could be used to modulate NER repair activities in vivo.

New Insights into Fluoroquinolone Resistance in Mycobacterium tuberculosis : Functional Genetic Analysis of gyrA and gyrB Mutations

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.

Kinetic Studies and Structural Models of the Association of E. coli σ70 RNA Polymerase with the λPR Promoter: Large Scale Conformational Changes in Forming the Kinetically Significant Intermediates

Abstract The kinetics of interaction of Eσ70 RNA polymerase (R) with the λPR promoter (P) were investigated by filter binding over a broad range of temperatures (7.3–42xa0°C) and concentrations of RNA polymerase (1–123xa0nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I2, RPo) are single-exponential with first order rate constant βCR. Interpretation of the polymerase concentration dependence of βCR in terms of the three step mechanism of open complex formation yields the equilibrium constant K1 for formation of the first kinetically significant intermediate (I1) and the forward rate constant (k2) for the conformational change converting I1 to the second kinetically significant intermediate I2: R + P ⇄ K 1 I 1 → k 2 I 2 . Use of rapid quench mixing allows K1 and k2 to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60xa0bp) interface formed in I1, its relatively small binding constant K1 at 37xa0°C at this [salt] (∼6×106xa0M−1) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of ka ( Δ C a o ,‡ =−1.5(±0.2) kcal K −1 ), now shown directly to originate largely from formation of I1 ( Δ C 1 o =−1.4(±0.3) kcal K −1 ), rather than from the formation of I2 as previously proposed. The isomerization I1→I2 exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy (E 2 act =34(±2) kcal ). This kinetic signature suggests that formation of the transition state (I1–I2)‡ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I1 involves coupled folding of unstructured regions of polymerase (β, β′ and σ70) and bending of promoter DNA (in the −10 region). We propose that interactions with region 2 of σ70 and possibly domain 1 of β induce a kink at the −11/−12 base pairs of the λPR promoter which places the downstream DNA (−5 to +20) in the jaws of the β and β′ subunits of polymerase in I1. These early interactions of β and β′ with the DNA downstream of position −5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.

Domain-swapped dimerization of the HIV-1 capsid C-terminal domain

Assembly of the HIV and other retroviruses is primarily driven by the oligomerization of the Gag polyprotein, the major viral structural protein capable of forming virus-like particles even in the absence of all other virally encoded components. Several critical determinants of Gag oligomerization are located in the C-terminal domain of the capsid protein (CA-CTD), which encompasses the most conserved segment in the highly variable Gag protein called the major homology region (MHR). The CA-CTD is thought to function as a dimerization module, although the existing model of CA-CTD dimerization does not readily explain why the conserved residues of the MHR are essential for retroviral assembly. Here we describe an x-ray structure of a distinct domain-swapped variant of the HIV-1 CA-CTD dimer stabilized by a single amino acid deletion. In the domain-swapped structure, the MHR-containing segment forms a major part of the dimerization interface, providing a structural mechanism for the enigmatic function of the MHR in HIV assembly. Our observations suggest that swapping of the MHR segments of adjacent Gag molecules may be a critical intermediate in retroviral assembly.

Unusual regioversatility of acetyltransferase Eis, a cause of drug resistance in XDR-TB

The emergence of multidrug-resistant and extensively drug-resistant (XDR) tuberculosis (TB) is a serious global threat. Aminoglycoside antibiotics are used as a last resort to treat XDR-TB. Resistance to the aminoglycoside kanamycin is a hallmark of XDR-TB. Here, we reveal the function and structure of the mycobacterial protein Eis responsible for resistance to kanamycin in a significant fraction of kanamycin-resistant Mycobacterium tuberculosis clinical isolates. We demonstrate that Eis has an unprecedented ability to acetylate multiple amines of many aminoglycosides. Structural and mutagenesis studies of Eis indicate that its acetylation mechanism is enabled by a complex tripartite fold that includes two general control non-derepressible 5 (GCN5)-related N-acetyltransferase regions. An intricate negatively charged substrate-binding pocket of Eis is a potential target of new antitubercular drugs expected to overcome aminoglycoside resistance.

The XPA-binding domain of ERCC1 Is Required for Nucleotide Excision Repair but Not Other DNA Repair Pathways

The endonuclease ERCC1-XPF incises the damaged strand of DNA 5′ to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1N110A/Y145A, the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1N110A/Y145A protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. We discuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin.

Analytic Binding Isotherms Describing Competitive Interactions of a Protein Ligand with Specific and Nonspecific Sites on the Same DNA Oligomer

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.

Organized Unidirectional Waves of ATP Hydrolysis within a RecA Filament

The RecA protein forms nucleoprotein filaments on DNA, and individual monomers within the filaments hydrolyze ATP. Assembly and disassembly of filaments are both unidirectional, occurring on opposite filament ends, with disassembly requiring ATP hydrolysis. When filaments form on duplex DNA, RecA protein exhibits a functional state comparable to the state observed during active DNA strand exchange. RecA filament state was monitored with a coupled spectrophotometric assay for ATP hydrolysis, with changes fit to a mathematical model for filament disassembly. At 37 °C, monomers within the RecA-double-stranded DNA (dsDNA) filaments hydrolyze ATP with an observed k cat of 20.8 ± 1.5 min−1. Under the same conditions, the rate of end-dependent filament disassembly (k off) is 123 ± 16 monomers per minute per filament end. This rate of disassembly requires a tight coupling of the ATP hydrolytic cycles of adjacent RecA monomers. The relationship of k cat to k off infers a filament state in which waves of ATP hydrolysis move unidirectionally through RecA filaments on dsDNA, with successive waves occurring at intervals of approximately six monomers. The waves move nearly synchronously, each one transiting from one monomer to the next every 0.5 s. The results reflect an organization of the ATPase activity that is unique in filamentous systems, and could be linked to a RecA motor function.

Survival in nuclear waste, extreme resistance, and potential applications gleaned from the genome sequence of Kineococcus radiotolerans SRS30216.

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energys Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible.