Atomistic Models from Orientation and Distance Constraints Using EPR of a Bifunctional Spin Label

Abstract

We have used high-resolution orientation and distance measurements derived from electron paramagnetic resonance of a bifunctional spin label (BSL) to build and refine atomistic models of protein structure. We demonstrate this approach by investigating the effects of nucleotide binding on the structure of myosin's catalytic domain while myosin is in complex with actin. Constraints for orientation of individual helices were obtained in a previous study from continuous-wave electron paramagnetic resonance of myosin labeled at specific sites with BSLs in oriented muscle fibers. In this study, new distance constraints were derived from double electron-electron resonance on myosin constructs labeled with a BSL specifically at two sites. Using these complementary constraints together, we thoroughly characterize the BSL's rigid, highly stereoselective attachment to protein a-helices, which permits accurate measurements of orientation and distance. We also leverage these measurements to derive a novel, to our knowledge, structural model for myosin-II in complex with actin and MgADP and compare our model to other recent actomyosin structures. The described approach is applicable to any orientable complex (e.g., membranes or filaments) in which site-specific di-Cys mutation is feasible.

Structural Dynamics of Protein Interactions Using Site-Directed Spin Labeling of Cysteines to Measure Distances and Rotational Dynamics with EPR Spectroscopy

“…Nevertheless, this enabled the engineering of cysteines for SDSL with monofunctional spin labels and bifunctional spin labels (BSL) to measure rotational motions of myosin S1 with STEPR [ 52 , 53 ] in the presence and absence of actin ( Fig. 11 ) and orientation changes in myosin segments [ 54 – 56 ] and in the LCD in muscle fibers [ 57 ]. BSL labeling adjacent cysteines acts like a crosslinker and was effective in cross-linking the actinbinding cleft of myosin [ 53 ] and SH1-SH2 cysteines in myosin [ 52 ] to capture elusive intermediate states ( Fig.…”

“…12a – c ). This orientation data was later combined with distance constraints from DEER experiments with BSL attached in same region to model the structure of myosin-II when bound to actin filaments [ 56 ] ( Fig. 12d – f ).…”

“…f Actin–myosin complex showing the helices in the apo and MgADP-bound myosin from modeling of measured distances. Figure is adapted from references with permission [ 54 , 56 ]…”