Determining interdomain structure and dynamics of a retroviral capsid protein in the presence of oligomerization: implication for structural transition in capsid assembly.

Large proteins (> 20 kDa) may consist of multiple domains. Individual domains can function as a scaffold or catalytic unit so that the full-length protein is more efficient in serving as a structural component or performing an enzymatic reaction, or both.(1) A retroviral capsid (CA) protein, consisting of two helical domains, is a typical example of the utilization of both domains to assemble a dense core to protect the retroviral genomic RNA in complex with the nucleocapsid (NC) protein. CA and NC proteins, in addition to matrix (MA) protein, are the three major proteolytic products of the precursor Gag poly-protein formed during viral maturation. CA proteins from all species of retroviruses are 26-27 kDa in size and structurally homologous.(2) X-ray crystallography and cryo-Electron-Microscopy (EM) structures of CA proteins from N-tropic murine leukaemia virus (N-MLV),(3) HIV-1,(4-6) and Rous Sarcoma Virus (RSV)(7) have revealed that the outer-layer of the capsid core is composed of a hexagonal and pentagonal assembly of the 7-helix CA N-terminal domain (CAN) and the inner-layer is formed through dimerization of neighboring hexamers by the 4-helix C-terminal domain (CAC). The inter-domain conformation within all known CA assembly is unique and most helices, helices α1-3 and α7 of the CAN and helices α8-11 of the CAC, are parallel to the hexamer symmetric axis. Though the inter-domain structure of CA is available for the assembled lattice, the assembly process of CA itself is dynamic and less understood.(8) The proteolysis cleavage of Gag poly-protein, a process that has been targeted by HIV-1 drug, regulates the release of CA, but may or may not be directly related to the CA assembly.(9, 10) Structure and dynamics studies of CA in solution can perhaps address a relevant question whether the assemble ready conformer has to be induced from or is already pre-existing in its monomer state in solution. Analogous to helix-coil transition(11) or protein-ligand conformational switch,(12) if a retroviral capsid assembly has to be induced, other factors, e.g. pH or ligand or protein-protein interaction, must be involved to create an activated conformer of CA from its equilibrium state, corresponding to the initial process of nucleation-propagation; in contrary, pre-existing would suggest the assembly starts from a sub-population of assemble ready inter-domain conformer within an equilibrated ensemble of CA structures in solution. Generally solution NMR can provide inter-domain structural information, i.e., inter-domain NOEs, residual dipolar coupling (RDC),(13-16) and relaxation time ratios T1/T2.(17-22) NOEs are measurable only if the inter-domain contacts involving protons are within a short distance and long lived. Even when these requirements are fulfilled, the NOEs may not be measureable in large proteins, since deuteration maybe required to obtain sharper resonance lines. RDCs are very useful in positioning domains relative to each other. If a multi-domain protein is within oligomerization equilibrium, RDCs are averaged results of all possible oligomers exchanging up to millisecond time scale and are strongly biased toward larger oligomers. RDC data interpretations quickly become difficult if the oligomerization is coupled to inter-domain structural change. The ratio of backbone 15N spin relaxation times T1/T2 is insensitive to fast local dynamics of the amide H-N bond,(23) and reflects the overall rotational diffusion of the domain.(24-27) The average T1/T2 values as well as their variation can be used to estimate a domain rotational correlation time and its geometrical shape, respectively. The average of each domain can be directly used to indicate the presence of inter-domain motion.(28, 29) The previous solution NMR studies on RSV-CA(28) have suggested that the motion of the two retroviral CA domains are independent of each other based on the lack of inter-domain NOEs and the difference in 15N T1/T2.(8) This observation is consistent with the pre-existing mechanism for CA assembly. However, CA proteins are likely to be in oligomerization equilibrium through domains CAN or CAC, and the measured T1/T2 might contain contributions from oligomers that can bias the relaxation values for each domain differently. For instance, the CA protein of Equine Infectious Anemia Virus (EIAV), belonging to the same lentivirus subfamily as HIV-1, weakly oligomerize and dimerize through its the CAN and CAC domains, respectively. And these two equilibriums appear to be independent processes as a result of the different motional time scales.(30) The assembled EIAV capsid is in the same conical shape as HIV-1 capsid.(31) The study here is to use NMR 15N relaxation and extrapolation data analysis to overcome oligomerization problem and at the same time to quantitatively reveal if the assemble-ready inter-domain conformer could be present in the conformational space of a monomeric EIAV-CA. Our results showed that the inter-domain structure of EIAV-CA monomer is relatively rigid and different from the assembled structure, and the CA assembly should be induced by other factors.