Solution Structure of a Double Mutant of the Carboxy-Terminal Dimerization Domain of the HIV-1 Capsid Protein^,

Abstract

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.

Structural and dynamical characterization of tubular HIV‐1 capsid protein assemblies by solid state nuclear magnetic resonance and electron microscopy

“…Detailed chemical shift assignments are not possible from the 2D spectrum in Figure 3(A) alone, of course. However, a qualitative comparison with the chemical shifts of NTD and CTD determined by solution NMR 23,27 can be performed. In Figure 3(B), a simulated 2D 13 CA 13 C spectrum is overlaid with the experimental spectrum, assuming crosspeaks only between the solution NMR chemical shifts of directly bonded 13 C sites and assuming the ideal residue-specific labeling patterns expected when 1,3-13 C 2 -glycerol is the sole carbon source.…”

“…By comparing DQ/SQ crosspeaks in Figure 5 with solution NMR chemical shifts for NTD and CTD, 23,27 111 crosspeaks were found to have unique assignments (see Table S1 of Supporting Information). Deviations of SQ 13 C NMR frequencies from the solution NMR chemical shift values were less than 0.5 ppm for 80 of these crosspeaks and less than 1.0 ppm for 16 crosspeaks.…”

“…1 The capsid is believed to be formed by a triangular lattice of CA hexamers, with 12 CA pentamers unevenly distributed at the two ends of the cone to produce a closed shell. 2 Although much progress has been made toward understanding the structure, stability, and assembly of the HIV-1 capsid, through electron microscopy, [3][4][5][6][7][8][9][10] X-ray crystallography, [11][12][13][14][15][16][17][18][19][20] solution nuclear magnetic resonance (NMR), 13,[21][22][23][24][25][26][27] and various biophysical techniques, [28][29][30][31][32][33][34][35][36][37][38][39][40] substantial questions remain regarding the details of intermolecular interactions within and between CA hexamers, the structural variations that permit CA to form both hexamers and pentamers, the driving force for curvature and closure of the CA lattice, and the mechanism and pathway for CA self-assembly. In principle, modern solid state NMR techniques, which can provide sitespecific molecular-level structural information about noncrystalline protein assemblies, [41][...…”

“…High-resolution structures of NTD and CTD have been determined by solution NMR and X-ray crystallography. [15][16][17][18][19][20][21][22][23][24]27,50 In these structures, NTD contains seven a-helical segments, plus a b-hairpin in the first 12 residues. CTD contains four a-helical segments, plus a single-turn 3 10 helix in the first 4 residues.…”

“…16,18,51 Monomeric CTD can be generated by mutation of certain residues in the dimer interface. 21,23 Recombinant wild-type CA self-assembles spontaneously into tubular structures at high ionic strength in vitro, 2,3,32,37,40 or in the presence of crowding agents. 29 Tubular CA assemblies are believed to be constructed from the same underlying hexamer lattice as the capsids of intact HIV-1, as supported by electron microscopy.…”