Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

Abstract

We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg 18 -Leu mutant (R18L-CA), and R18L-CA spheres with 20 -100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of 15 N, 13 C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in 15 N and 13 C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

Helical Conformation in the CA-SP1 Junction of the Immature HIV-1 Lattice Determined from Solid-State NMR of Virus-like Particles

“…In the mature lattice, residues 1–13 form a partially ordered β -hairpin, 15b with strong signals from residues 1–6 in ssNMR spectra of CA tubes. 11a These signals are absent from ssNMR spectra of VLPs, as expected if the covalent linkage to ΔMA prevents β -hairpin formation at the N-terminal segment of CA. On the other hand, the C-terminal segment of CA, which is invisible in ssNMR spectra of CA tubes, 11a becomes visible in VLPs, as discussed above.…”

“…10 ssNMR measurements of 15 N- 15 N dipole–dipole couplings indicate good agreement of backbone conformations at G220 and V230 between VLPs and the CTD-SP1 crystal structure (Figure S3). In contrast, residues 220–223 are dynamically disordered in tubular CA assemblies, 6c,11a unobserved in crystal structures of mature capsid constructs, 15 and disordered in structure bundles from solution NMR of CTD, CA, and full-length Gag. 16 However, backbone torsion angles predicted from chemical shifts of soluble, unassembled CA constructs (Figure S4) are similar to those obtained from our ssNMR data for VLPs.…”

“…ssNMR signals from these residues are entirely absent from corresponding ssNMR spectra of the mature lattice in CA assemblies, including tubular, spherical, and planar assemblies, due to dynamic disorder. 6c,11 …”

“…10 To identify additional evidence for structural rearrangements involving both NTD and CTD, we obtained partial chemical shift assignments for these domains from the 3D ssNMR spectra of ΔMA-CA-SP1-NC VLPs and compared them with our previously reported ssNMR data for tubular CA assemblies, which contain the mature lattice. 11a Assignments were facilitated by the flexibility of ΔMA and NC domains, which contribute few signals to the ssNMR spectra of VLPs, since the ssNMR measurement conditions favor immobilized segments. In cases where direct comparison of VLP spectra and CA tube spectra did not yield unambiguous assignments, due to large resonance shifts or spectral overlap, we used sequential correlations to verify the assignments.…”

Strategies for identifying dynamic regions in protein complexes: flexibility changes accompany methylation in chemotaxis receptor signaling states

“…Solid-state NMR is ideal for studies of large protein complexes and membrane proteins because it is not reliant on long range order, solubility, or rapid tumbling [5]. In the past decade, solid-state NMR has been used to study a variety of biological systems such as HIV-1 capsid protein assemblies [6][7][8][9][10][11], 300 kDa GB1-antibody complexes [12,13], and membrane-bound influenza M2 proton channels [14][15][16]. Such NMR studies have also provided measurements of protein dynamics [17][18][19] and identification of disordered regions [20][21][22][23][24].Research in recent years on intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) has highlighted the importance of disordered regions in the function of many proteins.…”