Methods to accelerate protein structure determination by solution NMR

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Structural analysis of meta-stable proteins is challenging because measured parameters often reflect a population weighted average over the different states dynamically sampled by such systems. Such a case applies for the membrane proximal external region (MPER) of HIV-1 gp41, which contains epitopes for at least four broadly neutralizing antibodies. Depending on solution conditions and construct design, different structures have been reported for this segment. We have applied novel methods to show that in aqueous solution the MPER fragment (gp160(660-674)) exists in a monomer-trimer equilibrium with an association constant in the micromolar range. Thermodynamic analysis revealed that the association is exothermic, more favorable in D2O than H2O, and increases with ionic strength. These observations indicate that the intermolecular interactions are hydrophobically driven. Circular dichroism, C-13(alpha) chemical shifts, NOE, and hydrogen exchange rates revealed that MPER undergoes a structural transition from predominately unfolded monomer at low concentrations to an alpha-helical trimer at high concentrations. This result has implications for antibody recognition of MPER prior to and during the process where gp41 switches from a pre-hairpin intermediate to its post-fusion 6-helical bundle state. A series of novel three- and four-dimensional NMR experiments has been developed that provide access to the structure and dynamics of the 608-residue homodimeric Main protease of the SARS-CoV-2 virus. This protein is larger than what is readily accessible to the standard NMR approaches used, but the novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural analysis which reveals subtle but statistically very significant differences relative to all X-ray structures available so far. A characterization of the backbone dynamics shows a strong effect of ligands on the dynamics of the protein backbone in the active site region. Alternative procedures to acquire and process triple-resonance data for measurement of residual dipolar couplings (RDCs) in combination with non-uniform sampling have been developed that increase the precision at which such RDCs can be extracted from the data. The TROSY-HNCO 3D NMR experiments was extended in a manner similar to that of the original amplitude-modulated RDC TROSY Spectroscopy (ARTSY) experiment to obtain vastly improved spectral resolution, which allowed the measurement of a nearly complete set of data for the SARS-CoV-2 main protease, and to assess its structure in solution and to compare it with the many X-ray structures deposited in the protein databank (PDB). Results show that although, on average, solution measurements are in good agreement with the X-ray data, in several localized regions the solution data fall outside the range observed in the nearly 200 X-ray structures. Substantial differences appear in the active site region, and refinement of the solution NMR structure of this homodimeric structure are currently in progress.