Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins

Abstract

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16), a complex essential for virus survival in the host and thus a desirable drug target. Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with the literature, well characterized small cosubstrate, RNA substrate, and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound cosubstrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, we observed an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation. The metal migration can be suppressed by using surface collision with reduced precursor charge states, which presumably resulted in minimal gas-phase structural rearrangement and highlighted the importance of complementary techniques. With minimal sample input (∼μg), native MS can rapidly detect ligand binding affinities and locations in dynamic multisubunit protein complexes, demonstrating the potential of an "all-in-one" native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.

Figure 1

(A) Native MS spectrum showing 10 μM of a mixture of nsp10, nsp16, and the heterodimer. The inset shows the magnified display of the dimer peaks. (B) Dimer formation can be directly tracked by the changing intensities as a function of protein concentration. (C) A different construct of nsp10’ (blue trace) has 6.9 Da lower mass and extra terminal clipping peaks from the original nsp10 (black trace). (D) Mixing ∼33 μM of the nsp10’ with 10 μM of the original nsp10/16 resulted in subunit exchange in the dimer, as manifested by the mass shift in the dimer peak (black to blue trace).

Figure 2

Deconvolved mass spectra for 6 μM SAM binding to (A) nsp10, (B) nsp16, and (C) nsp10/16 complex. The heat maps (D) show the percentage of the total peak area that is ligand-bound for each species as the ligand concentration was increased from 0–20 μM for SAM and SAH and from 0–60 μM for SFG. Estimated K_d curves (E) are shown for each ligand when fit to a single-site binding model.

Figure 3

Deconvolved mass spectra of nsp10, nsp16, and nsp10/16 at 20 μM added ligand. (A) Binding of the designed compound was nonspecific, indiscriminately binding to the dimer and monomers. (B) In contrast, SAM displayed negligible binding to monomers but specific and significant binding to the dimer, with a lower but distinct second SAM binding event clearly visible. Like SAM, SAH (C) and SFG (D) also predominantly bind to the dimer and display a second peak corresponding to two bound ligands. However, unlike SAM, the small ligand peaks in the monomer spectra indicate that some degree of nonspecific binding occurs.

Figure 4

(A) Nsp10/16 was isolated from monomers and subjected to collisional dissociation in 2 V increments from 4 to 60 V. (B) Peak areas were extracted as a function of collision voltage and plotted for SAM-bound nsp10/16 (pink triangles), apo nsp10/16 (blue circles), SAM-bound nsp16 (green triangles), apo nsp16 (black circles), SAM-bound nsp10 (orange triangles), and apo nsp10 (purple circles). Additional peaks visible in the nsp10 and nsp16 spectra (A) are due to either 1 or 2 bound Zn ions.

Figure 5

Deconvolved intact mass spectra of nsp10 (left column) and nsp16 (right column) monomers either as (A–B) free monomers in solution or (C–D) released from the heterodimer after dissociation. Data from CID are in red traces, and SID are in blue traces. Zn-bound stoichiometry is annotated above the peaks. Charge reduced condition with TEAA addition are in (E–H) following the same format. For the nsp10/16 dimer at 13+, both SID and CID resulted in similar levels of Zn transfer. TEAA did not affect the bound Zn on free nsp16 in solution prior to dissociation, but the Zn transfer was largely eliminated in SID after charge reduction while CID tends to induce a higher degree of Zn transfer.