As in other retroviruses, the HIV-1 capsid (CA) protein is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), joined by a flexible linker. The dimerization of the CTD is thought to be a critical step in the assembly of the immature and mature viral capsids. The precise nature of the functional form of CTD dimerization interface has been a subject of considerable interest. Previously, the CTD dimer was thought to involve a face-to-face dimerization observed in the early crystallographic studies. Recently, the crystallographic structure for a domain-swapped CTD dimer has been determined. This dimer, with an entirely different interface that includes the major homology region (MHR) has been suggested as the functional form during the Gag assembly. The structure determination of the monomeric wt CTD of HIV-1 has not been possible because of the monomer-dimer equilibrium in solution. We report the NMR structure of the [W184A/M185A]-CTD mutant in its monomeric form. These mutations interfere with dimerization without abrogating the assembly activity of Gag and CA. The NMR structure shows some important differences compared to the CTD structure in the face-to-face dimer. Notably, the helix-2 is much shorter, and the kink seen in the crystal structure of the wt CTD in the face-to-face dimer is absent. These NMR studies suggest that dimerization-induced conformational changes may be present in the two crystal structures of the CTD dimers and also suggest a mechanism that can simultaneously accommodate both of the distinctly different dimer models playing functional roles during the Gag assembly of the immature capsids.
DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP-bound conformation and high binding affinity for dGTP-Mg(2+) of Pol X may contribute to its low fidelity.
The capsid protein (CA) of the HIV-1 virus plays a significant role in the assembly of the immature virion, and is the critical building block of its mature capsid. Thus, there has been a significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 virus replication cycle. However, due to its inherent flexibility from the inter-domain linker and the monomer-dimer equilibrium in solution, HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and NMR spectroscopy. Here we report the detailed solution structure of the full length HIV-1 CA using a monomeric mutant that, though non-infective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD domain shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by the HIV-1 virus, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of the HIV-1 virus.
The surprising observation that a 10 residue class G* peptide from apolipoprotein J, [113-122]apoJ, possesses anti-inflammatory and anti-atherogenic properties prompted us to delineate its structural characteristics in the presence of normal and oxidized lipid. Towards this, we have determined high resolution structure of [113-122]apoJ in solution using nuclear magnetic resonance (NMR) spectroscopy and studied its interaction with lipids, including oxidized lipids, using a number of biophysical methods. Circular dichroism and NMR studies established that in the presence of dodecylphosphocholine (DPC) micelle this peptide adopts amphipathic α helical structure. The observed Nuclear Overhauser effects indicate that the amphipathic helical structure of the peptide is stabilized by the N-terminal acetyl and C-terminal amide blocking groups. We used isothermal titration calorimetry to measure binding enthalpy of the peptide with DPC micelle, an oxidized lipid, 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdiA-PC), and the mixture of these two lipids (5mol% KOdiA-PC in DPC micelle). We find that the peptide binding with DPC micelle is associated with an enthalpy change (-16.75±0.16 Kcal/mol) much larger than that resulting from the binding with KodiA-PC (-3.67±0.13 Kcal/mol). Incorporation of a small amount of KOdiA-PC (5mol %) in DPC micelle also results in the lowering of peptide binding enthalpy (-13.43±0.18 Kcal/mol). These results are consistent with overall negative charge and altered conformational properties of oxidized sn-2 chain of KOdiA-PC. Our results have unambiguously established the amphipathic α helical structure of [113-122]apoJ peptide in the presence of DPC micelle as well as its ability to bind oxidized lipid. These in vitro results help explain the previously observed anti-inflammatory and anti-atherosclerotic properties of this peptide.
A careful computer analysis of the location of positively charged residues in the tandem amphipathic helical domains of human apoA-I shows the preponderance of arginine (Arg) residues on the right hand side of the polar face of the amphipathic helix ( 1 ). Because HDL particles containing apoA-I are major carriers of antioxidant enzymes ( 2 ), and the analysis of putative HDL-associating regions of paraoxonase 1 (PON1) showed the presence of clusters of aromatic amino acids ( 3 ), we hypothesized that the side-specifi c Arg residues in human apoA-I are involved in cation-interactions with the aromatic residues present in the putative HDL binding region of PON1 ( 1 ).We have shown that the interfacial lysine (Lys) residues in the class A amphipathic helical peptide 2F are in different microenvironments with sidedness in their pKa values; Lys residues located on the left hand side have lower pKa value than the Lys residues located on the right hand side of the amphipathic helix ( 4 ). In addition, we have shown that the apoA-I mimetic peptides are arranged in discoidal complexes in a head-to-tail manner in which the Lys residues with higher pKa values likely interact with phosphate in the lipid head group and Lys residues possessing lower pKa values are present in a more hydrophobic environment ( 5 ). Because Arg residues in apoA-I are predicted to be involved in cation-interactions with PON1 ( 1 ), we tested the hypothesis that side-specifi c incorporation of Arg residues in the apoA-I mimetic peptide 4F results in peptides that exhibit differential biological properties. Thus, we synthesized (as shown in Fig. 1 ) two additional analogs of 4F, a peptide that has been extensively studied for its various anti-infl ammatory properties by us as well as
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