Unusual Polymorphisms in Human Immunodeficiency Virus Type 1 Associated with Nonprogressive Infection

Factors accounting for long-term nonprogression may include infection with an attenuated strain of human immunodeficiency virus type 1 (HIV-1), genetic polymorphisms in the host, and virus-specific immune responses. In this study, we examined eight individuals with nonprogressing or slowly progressing HIV-1 infection, none of whom were homozygous for host-specific polymorphisms (CCR5-Δ32, CCR2-64I, and SDF-1-3\u27A) which have been associated with slower disease progression. HIV-1 was recovered from seven of the eight, and recovered virus was used for sequencing the full-length HIV-1 genome; full-length HIV-1 genome sequences from the eighth were determined following amplification of viral sequences directly from peripheral blood mononuclear cells (PBMC). Longitudinal studies of one individual with HIV-1 that consistently exhibited a slow/low growth phenotype revealed a single amino acid deletion in a conserved region of the gp41 transmembrane protein that was not seen in any of 131 envelope sequences in the Los Alamos HIV-1 sequence database. Genetic analysis also revealed that five of the eight individuals harbored HIV-1 with unusual 1- or 2-amino-acid deletions in the Gag sequence compared to subgroup B Gag consensus sequences. These deletions in Gag have either never been observed previously or are extremely rare in the database. Three individuals had deletions in Nef, and one had a 4-amino-acid insertion in Vpu. The unusual polymorphisms in Gag, Env, and Nef described here were also found in stored PBMC samples taken 3 to 11 years prior to, or in one case 4 years subsequent to, the time of sampling for the original sequencing. In all, seven of the eight individuals exhibited one or more unusual polymorphisms; a total of 13 unusual polymorphisms were documented in these seven individuals. These polymorphisms may have been present from the time of initial infection or may have appeared in response to immune surveillance or other selective pressures. Our results indicate that unusual, difficult-to-revert polymorphisms in HIV-1 can be found associated with slow progression or nonprogression in a majority of such cases

Effect of Stalling after Mismatches on the Error Catastrophe in Nonenzymatic Nucleic Acid Replication

The frequency of errors during genome replication limits the amount of functionally important information that can be passed on from generation to generation. During the origin of life, mutation rates are thought to have been quite high, raising a classic chicken-and-egg paradox: could nonenzymatic replication propagate sequences accurately enough to allow for the emergence of heritable function? Here we show that the theoretical limit on genomic information content may increase substantially as a consequence of dramatically slowed polymerization after mismatches. As a result of postmismatch stalling, accurate copies of a template tend to be completed more rapidly than mutant copies and the accurate copies can therefore begin a second round of replication more quickly. To quantify this effect, we characterized an experimental model of nonenzymatic, template-directed nucleic acid polymerization. We found that most mismatches decrease the rate of primer extension by more than 2 orders of magnitude relative to a matched (Watson-Crick) control. A chemical replication system with this property would be able to propagate sequences long enough to have function. Our study suggests that the emergence of functional sequences during the origin of life would be possible even in the face of the high intrinsic error rates of chemical replication

Mutational Specificity of γ-Radiation-Induced Guanine−Thymine and Thymine−Guanine Intrastrand Cross-Links in Mammalian Cells and Translesion Synthesis Past the Guanine−Thymine Lesion by Human DNA Polymerase η†

ABSTRACT: Comparative mutagenesis of γ- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8 % of progeny contained targeted base substitutions, whereas 10.0 % showed semitargeted single-base substitutions. Of the targeted mutations, the G f T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5 ′ and three bases 3 ′ to the cross-link. The most prevalent semitargeted mutation was a C f T transition immediately 5 ′ to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of ∼16%, and both included a dominant G f T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5 ′ to the lesion was increased and 3 ′ to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called “A-rule”. To determin

Conformational Entropy from Mobile Bond Vectors in Proteins: A Viewpoint that Unifies NMR Relaxation Theory and Molecular Dynamics Simulation Approaches

A new method for determining conformational entropy in proteins is reported. Proteins prevail as conformational ensembles, p - exp(-u). By selecting a bond vector (e.g., N-H) as a conformation representative, molecular dynamics simulations can provide (relative to a reference structure) p as approximate Boltzmann probability density and u as N-H potential of mean force (POMF). The latter is as accurate as implied by the force field but statistical in character; this limits the insights it can provide and its utilization. Conformational entropy is given exclusively by u. Deriving it from POMFs renders it accurate but statistical in character. Previously, we devised explicit (i.e., analytical but not exact) potentials made of Wigner functions, D0KL, with L ≤ 4, which closely resemble the corresponding POMFs in form; hence, they also approach the latter in accuracy. Such potentials can be beneficially characterized/compared in terms of composition, symmetry, and associated order parameters. In this study, we develop a method for deriving conformational entropy from them, which also features the benefits specified above. The method developed is applied to the dimerization of the Rho GTPase-binding domain of plexin-B1. Insights into local ordering, entropy compensation, and features of allostery are gained. In previous work, we developed the slowly relaxing local structure (SRLS) approach for the analysis of NMR relaxation from restricted bond vector motion in proteins. SRLS comprises explicit (restricting) potentials of the kind developed here. It also comprises diffusion tensors describing the local motion and related features of local geometry. The complete model fits experimental data. In future work, the explicit potentials developed here will be inserted unchanged in SRLS-based data fitting, thereby improving the picture of structural dynamics. Given that SRLS is unique in featuring potentials that can closely approach the corresponding POMFs in accuracy, the present study is an important step toward generally improving protein dynamics by NMR relaxation

Local Ordering at the N-H Sites of the Rho GTPase Binding Domain of Plexin-B1: Impact of Dimerization

We have developed a new molecular dynamics (MD) based method for describing analytically local potentials at mobile N-H sites in proteins. Here we apply it to the monomer and dimer of the Rho GTPase binding domain (RBD) of the transmembrane receptor plexin-B1 to gain insight into dimerization, which can compete with Rho GTPase binding. In our method, the local potential is given by linear combinations, u(Djavax.xml.bind.JAXBElement@314a08db), of the real combinations of the Wigner rotation matrix elements, DL,K, with L = 1-4 and appropriate symmetry. The combination that "fits best" the corresponding MD potential of mean force, u(MD), is the potential we are seeking, u(Djavax.xml.bind.JAXBElement@b86fbd8 - BEST). For practical reasons the fitting process involves probability distributions, Peq 1d exp(-u), instead of potentials, u. The symmetry of the potential, u(Djavax.xml.bind.JAXBElement@63b7e142), may be related to the irreducible representations of the D2h point group. The monomer (dimer) potentials have mostly Ag and B2u (B1u and B2u) symmetry. For the monomer, the associated probability distributions are generally dispersed in space, shallow, and centered at the "reference N-H orientation" (defined in section 3.1. below); for the dimer many are more concentrated, deep and centered away from the "reference N-H orientation". The u(Djavax.xml.bind.JAXBElement@7f761679) functions provide a consistent description of the potential energy landscape at protein N-H sites. The L1-loop of the plexin-B1 RBD is not seen in the crystal structure, and many resonances of the L4 loop are missing in the NMR 15N-1H HSQC spectrum of the dimer; we suggest reasons for these features. An allosteric signal transmission pathway was reported previously for the monomer. We find that it has shallow N-H potentials at its ends, which become deeper as one proceeds toward the middle, complementing structurally the previously derived dynamic picture. Prospects of this study include correlating u(Djavax.xml.bind.JAXBElement@1e1bc59c - BEST) with MD force-fields, and using them without further adjustment in NMR relaxation analysis schemes

Microsecond MD Simulations of the Plexin-B1 RBD: 2. N-H Probability Densities and Conformational Entropy in Ligand-Free, Rac1-Bound, and Dimer RBD

Orientational probability densities, Peq = exp(-u) (u, local potential), of bond-vectors in proteins provide information on structural flexibility. The related conformational entropy, Sk = -integral P-eq(ln P-eq)d omega - ln integral d omega, provides the entropic contribution to the free energy of the physical/biological process studied. We have developed a new method for deriving Peq and Sk from MD simulations, using the N-H bond as probe. Recently we used it to study the dimerization of the Rho GTPase binding domain of Plexin-B1 (RBD). Here we use it to study RBD binding to the small GTPase Rac1. In both cases 1 mu s MD simulations have been employed. The RBD has the ubiquitin fold with four mostly long loops. L3 is associated with GTPase binding, L4 with RBD dimerization, L2 participates in interdomain interactions, and L1 has not been associated with function. We find that RBD-Rac1 binding renders L1, L3, and L4 more rigid and the turns beta(2)/alpha(1) and alpha(2)/beta(5) more flexible. By comparison, RBD dimerization renders L4 more rigid, and the alpha-helices, the beta-strands, and L2 more flexible. The rigidity of L1 in RBDRAC is consistent with L1-L3 contacts seen in previous MD simulations. The analysis of the L3-loop reveals two states of distinct flexibility which we associate with involvement in slow conformational exchange processes differing in their rates. Overall, the N-H bonds make an unfavorable entropic contribution of (5.9 +/- 0.9) kJ/mol to the free energy of RBD-Rac1 binding; they were found to make a favorably contribution of (-7.0 +/- 0.7) kJ/mol to the free energy of RBD dimerization. In summary, the present study provides a new perspective on the impact of Rac1 binding and dimerization on the flexibility characteristics of the RBD. Further studies are stimulated by the results of this work

Microsecond MD Simulations of the Plexin-B1 RBD: N-H Probability Density as Descriptor of Structural Dynamics, Dimerization-Related Conformational Entropy, and Transient Dimer Asymmetry

Amide-bond equilibrium probability density, Peq = exp(-u) (u, local potential), and associated conformational entropy, Sk = -∫Peq (ln Peq) dω ln ∫dω, are derived for the Rho GTPase binding domain of Plexin-B1 (RBD) as monomer and dimer from 1 μs MD simulations. The objective is to elucidate the effect of dimerization on the dynamic structure of the RBD. Dispersed (peaked) Peq functions indicate "flexibility"("rigidity"the respective concepts are used below in this context). The L1 and L3 loops are throughout highly flexible, the L2 loop and the secondary structure elements are generally rigid, and the L4 loop is flexible in the monomer and rigid in the dimer. Overall, many residues are more flexible in the dimer. These features, and their implications, are discussed. Unexpectedly, we find that monomer unit 1 of the dimer (in short, d1) is unusually flexible, whereas monomer unit 2 (in short, d2) is as rigid as the RBD monomer. This is revealed due to their engagement in slow-to-intermediate conformational exchange detected previously by 15N relaxation experiments. Such motions occur with rates on the order of 103-104 s-1 hence, they cannot be completely sampled over the course of 1 μs simulation. However, the extent to which rigid d2 is affected is small enough to enable physically relevant analysis. The entropy difference between d2 and the monomer yields an entropic contribution of -7 ± 0.7 kJ/mol to the free energy of RBD dimerization. In previous work aimed at similar objectives we used 50-100 ns MD simulations. Those results and the present result differ considerably. In summary, bond-vector Peq functions derived directly from long MD simulations are useful descriptors of protein structural dynamics and provide accurate conformational entropy. Within the scope of slow conformational exchange, they can be useful, even in the presence of incomplete sampling

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (K(i)) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a K(i) of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with K(i) of 1–4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys(21) and Tyr(22), are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg(23), indicating that Lys(21) is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors