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. New three- and four-dimensional NMR experiments have been developed that provide access to the structure and dynamics of systems that are large in comparison to proteins normally studied by NMR spectroscopy, such as the homodimeric Main protease of the SARS-CoV-2 virus. The novel methods combined with 900 MHz high-field measurements provided sufficient data for a detailed structural characterization that reveals subtle but statistically quite significant differences relative to all X-ray structures available so far. A characterization of the backbone motions shows a strong effect of substrate binding on the dynamics of the protein backbone in the active site region. Specifically, new schemes 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 indicates that many of the residues near the active site exist in a microsecond timescale equilibrium between multiple states. None of the X-ray structures, provides a good fit to the solution NMR RDCs, although some improvement in agreement is observed when comparing the NMR data to ensemble models developed by the PHENIX method to fit X-ray diffraction data to ensembles of structures. Fitting of residual dipolar couplings (RDCs) to proteins of known structure frequently yields poor fits during the early rounds of such analysis, making it difficult or impossible to obtain adequate estimates for the alignment tensor. We demonstrate for three proteins, maltotriose-ligated maltose binding protein (MBP), Ca2+-ligated calmodulin, and a monomeric N-terminal deletion mutant of the SARS-CoV-2 Main Protease, Mpro, that accurate alignment tensors of their domains can be obtained from RDCs that are measured in an aqueous solution containing a dilute liquid crystalline suspension of filamentous virus Pf1. The program, Helix-fit, fits the RDCs to idealized alpha-helical coordinates which for calmodulin and MBP yields a comparable or better alignment tensor estimate than fitting to the actual high-resolution X-ray helix coordinates. Helix-fit provides access to domain dynamics before an actual structure is known. The nine helices of ligated MBP show very similar alignment tensors, indicative of a high degree of order relative to one another. By contrast, alignments of the helices in the N- and C-domains of Ca2+- calmodulin differ from one another, confirming previously identified domain motions. For monomeric Mpro, alignments of the six helices in the C-terminal domain are very similar, pointing to a well-ordered domain, but the single alpha-helix Y54-I59 in the N-terminal domain aligns considerably weaker, suggesting large amplitude motions of the C-terminal relative to the N-terminal domain. 3D printing of NMR sample cells was demonstrated to be of high practical utility for reducing the amount of sample required for NMR measurements by nearly two-fold. By printing the sample cell in an ellipsoidal shape, magnetic susceptibility mismatch problems are avoided and good lineshapes are obtained. Unlike conventional NMR sample tubes, the 3D printed sample cells are compatible with high ionic strength, allowing the recording of high quality spectra at salt concentrations of at least 2 M. The new sample cells are orders of magnitude less expensive than commercial alternatives, and require less sample to be prepared. The printing costs of the cells are negligible (<$1), and the savings in time and isotopes needed for NMR experiments is at least two-fold.