Modelling multi-protein complexes using PELDOR distance measurements for rigid body minimisation experiments using XPLOR-NIH

Abstract

Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model.

The contribution of modern EPR to structural biology

“…Such problems can be overdetermined by distance distribution restraints. The two-body problem can be solved by a genetic algorithm implemented in mtsslSuite [32], by a complete grid search [23,26], or by an Xplor-NIH [47]-based protocol [48]. For more than two bodies, the RigiFlex approach has been proposed [26].…”

“…The Xplor-NIH-based protocol for determining relative domain orientation and translation in proteins with two domains [48], developed in a pioneering study on a tandem of polypeptide transport-associated domains in Omp85 [54], integrates EPR distances with NMR nuclear Overhauser Effect (NOE) restraints. The structure was still ill-defined with 2907 short-range NOE restraints and could be fixed by only three additional DEER restraints.…”

Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics

“…[235] By using a modified AlphaFold approach, diverse conformational states of transporter proteins were first generated, [235] which were further validated through systematic PDS-EPR measurements. [236] Xplor-NIH and MELD with PDS-EPR: Other emerging simulation tools have incorporated EPR distance restraints to better sample protein conformations, including Xplor-NIH [237] and modeling employing limited data (MELD). [238] Xplor-NIH was initially designed to automatically interpret raw NMR signals into molecular structures.…”

“…[239] In recent years, incorporation of EPR distance restraints obtained between interacting proteins in Xplor-NIH has enabled the prediction of the relative orientations of a multi-protein complex, such as the histone chaperone Vps75. [237] Alternatively, a free-energy-based computational method, MELD, determined protein structures using a Bayesian approach combined with limited experimental data. [239] The MELD approach utilizes free energy as criteria to choose conformational states that possibly correspond to the sparse experimental data.…”

“…Xplor‐NIH and MELD with PDS‐EPR : Other emerging simulation tools have incorporated EPR distance restraints to better sample protein conformations, including Xplor‐NIH [237] and modeling employing limited data (MELD) [238] . Xplor‐NIH was initially designed to automatically interpret raw NMR signals into molecular structures [239] .…”