Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking

Abstract

Structural determination of protein-protein complexes such as multidomain nuclear receptors has been challenging for high-resolution structural techniques. Here, we present a combined use of multiple biophysical methods, termed iSPOT, an integration of shape information from small-angle X-ray scattering (SAXS), protection factors probed by hydroxyl radical footprinting, and a large series of computationally docked conformations from rigid-body or molecular dynamics (MD) simulations. Specifically tested on two model systems, the power of iSPOT is demonstrated to accurately predict the structures of a large protein-protein complex (TGFβ-FKBP12) and a multidomain nuclear receptor homodimer (HNF-4α), based on the structures of individual components of the complexes. Although neither SAXS nor footprinting alone can yield an unambiguous picture for each complex, the combination of both, seamlessly integrated in iSPOT, narrows down the best-fit structures that are about 3.3 Å and 4.2 Å in RMSD from their corresponding crystal structures, respectively. Furthermore, this proof-of-principle study based on the data synthetically derived from available crystal structures shows that the iSPOT—using either rigid-body or MD-based flexible docking—is capable of overcoming the shortcomings of standalone computational methods, especially for HNF-4α. By taking advantage of the integration of SAXS-based shape information and footprinting-based protection/accessibility as well as computational docking, this iSPOT platform is set to be a powerful approach towards accurate integrated modeling of many challenging multiprotein complexes.

A metastable contact and structural disorder in the estrogen receptor transactivation domain

“…Here, we supplement SAXS data by hydroxyl radical protein footprinting (FP) coupled with mass spectrometry, where transforming the data to protection factors (PFs) can report solvent accessibility of residue side chains. The value of this approach in multi-technique modeling protein structures and protein-protein interactions has been demonstrated in our previous proof-of-principle studies (Huang et al, 2016) and more recently in the structure determination of a multidomain estrogen receptor complex . To measure FP-PF data, purified NTD samples were exposed to a focused synchrotron X-ray white beam, where radiolysis-generated hydroxyl radicals react with solvent accessible side chains, and the sites and rates of oxidation are monitored by tandem mass spectrometry MS (Takamoto and Chance, 2006).…”

“…where I cal is calculated using fast-SAXS-pro (Ravikumar et al, 2013) and N q is the number of scattering q points recorded in experimental I exp (with its measurement error of σ(q)). Similarly, the φ 2 is the goodness of fit between experimental and theoretical FP-FP data by (Hsieh et al, 2017;Huang et al, 2016 where SA cal is the predicted side-chain SA value of each site, using the linear regression between experimental logPF exp values (with an error δ(s) at each site (s)) and SA values of N s sites for each structural cluster (here, N s =16 as shown in Table S3).…”

“…Dash lines were used for visualization only. Oligonucleotides S118D-FWD:5'-CGCCGCAGCTGGACCCTTTCCTGCAG-3' S118D-REV:5'-CTGCAGGAAAGGGTCCAGCTGCGGCG-3' S118C FWD:5'-CCGCCGCAGCTGTGCCCTTTCCTGCAGC-3' S118C REV:5'-GCTGCAGGAAAGGGCACAGCTGCGGCGG-3' L31A FWD:5'-tgaaccgtccgcaggccaagatccccctgg-3' L31A REV:5'-ccagggggatcttggcctgcggacggttca-3' K32A,I33A,P34A FWD: 5'-aaccgtccgcagctcgcggccgccctggagcggcccc-3' K32A,I33A,P34A REV: 5'-ggggccgctccagggcggccgcgagctgcggacggtt-3' (Huang et al, 2016) http://www.theyanglab.org/ispot PF-FP (Huang et al, 2015) http://www.theyanglab.org/protection.html…”

“…Hence, the accuracy of the force field used is beyond the scope here, and these conformations were combined and selected by comparison with multi-technique experimental measurements. To group similar structures together and reduce the computational cost, we performed an RMSDbased clustering analysis as previously described (Huang et al, 2016;Svergun, 1992). Using a Ca-RMSD cutoff of 5 Å, we obtained a set of N=8,491 clusters of NTD structures from the total of 35,240 simulation snapshots.…”

Molecular Dissection of the Intrinsically Disordered Estrogen Receptor Alpha-NTD

“…SAXS data was the source of size and shape information, while hydroxyl radical footprinting gave a measure of side chain solvent accessibility. Together, SAXS and the footprinting provide complementary information and this has previously been used to probe the structure of oligomers (Wang et al, 2011), protein complexes (Huang et al, 2016), and multidomain proteins (Huang et al, 2018). However, since ERa-NTD is an IDP, both SAXS and hydroxyl radical footprinting yielded information that described the ensemble average, rather than a single model.…”