Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State.

Reza Amani,Benjamin J Wylie,Ruixian Han,Charles D Schwieters,Benjamin D Harding,Isaac R Eason,Collin G Borcik

doi:10.3389/fmolb.2021.772855

Abstract

NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1–300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

Highlights

Solid-state NMR (SSNMR) is essential to the structural and functional characterization of membrane proteins (MPs) (Schubeis et al, 2018; Radoicic et al, 2014; Wylie et al, 2016; Mandala et al, 2018; Tran et al, 2020)
Following observations reported by several groups and within our own laboratory, we found that large domain motions may be mapped by the observable solvent accessible surface (Borcik et al, 2020)
The statistical comparison of the 10 lowest energy structures solved with water-edited SSNMR restraints supplied to the PSolPot potential improved both the backbone and all heavy-atom RMSDs relative to ensembles without these restraints

Summary

Introduction

Solid-state NMR (SSNMR) is essential to the structural and functional characterization of membrane proteins (MPs) (Schubeis et al, 2018; Radoicic et al, 2014; Wylie et al, 2016; Mandala et al, 2018; Tran et al, 2020). Over the past two decades the water accessible surface of MPs was actively quantified via SSNMR (Kumashiro et al, 1998; Ader et al, 2009; Li et al, 2010; Su et al, 2011; Hornig et al, 2013). Over this time, water-edited SSNMR examined the rearrangement of membrane proteins, molecular motion in deuterated samples, and determined membrane insertion topology (Najbauer et al, 2019). In pursuit of functional states of K+ channels, Ader et al used water-edited SSNMR spectroscopy to unambiguously uncover a dramatic

Methods

Results

Conclusion