Activation of Nanoscale Allosteric Protein Domain Motion Revealed by Neutron Spin Echo Spectroscopy

Abstract

NHERF1 is a multidomain scaffolding protein that assembles signaling complexes, and regulates the cell surface expression and endocytic recycling of a variety of membrane proteins. The ability of the two PDZ domains in NHERF1 to assemble protein complexes is allosterically modulated by the membrane-cytoskeleton linker protein ezrin, whose binding site is located as far as 110 Ångstroms away from the PDZ domains. Here, using neutron spin echo (NSE) spectroscopy, selective deuterium labeling, and theoretical analyses, we reveal the activation of interdomain motion in NHERF1 on nanometer length-scales and on submicrosecond timescales upon forming a complex with ezrin. We show that a much-simplified coarse-grained model suffices to describe interdomain motion of a multidomain protein or protein complex. We expect that future NSE experiments will benefit by exploiting our approach of selective deuteration to resolve the specific domain motions of interest from a plethora of global translational and rotational motions. Our results demonstrate that the dynamic propagation of allosteric signals to distal sites involves changes in long-range coupled domain motions on submicrosecond timescales, and that these coupled motions can be distinguished and characterized by NSE.

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

“…Also, there is increasing evidence that the activated ezrin binds to target proteins that trigger the subsequent propagation of downstream allosteric binding signals in the membrane cytoskeleton (15,80,81). For instance, ezrin activates NHERF1 and induces long range allostery in NHERF1 so as to strengthen the interactions of NHERF1 with transmembrane proteins and other signaling proteins (24,27,70). In turn, NHERF1 also allosterically activates other proteins, such as the scaffolding protein PDZK1 that binds to downstream targets for the assembly microvillus structures on the cell surface (15,81).…”

“…In turn, NHERF1 also allosterically activates other proteins, such as the scaffolding protein PDZK1 that binds to downstream targets for the assembly microvillus structures on the cell surface (15,81). Ezrin is a crucial player in protein dynamics, long range allostery, and signal transduction (70,82).…”

“…This is likely due to swivel-like motions between the FERM and C-ERMAD about the hinge connecting the two halves of the central helices. Previously, we have shown the highly fluctuating region of a protein tends to be overestimated by the ab initio reconstruction method (70). S5, A and B).…”

Ligand-Induced Dynamic Changes in Extended PDZ Domains from NHERF1

“…Association of Ezrin releases the autoinhibited conformation from intra-molecular head-to-tail interactions between PDZ2 and the carboxy-terminal PDZ binding motif in EBD 11; 12; 13; 14; 15; 16 . The bivalent NHERF1 is active predominantly in trafficking and function of a number of membrane proteins, including ion channels 7 and GPCR coupled receptors 17; 18; 19 facilitated through association with ezrin and other ERM (ezrin-radixin-moesin) proteins from the actin cytoskeleton 20 .…”

Thermal activation of ‘allosteric-like’ large-scale motions in a eukaryotic Lactate Dehydrogenase

“…Particularly, elastic, quasielastic, and inelastic incoherent NS have been exploited not only to study the sub-nanosecond timescale local functional dynamics of model proteins1819 and their solvent2021, but also for in-vivo investigations of bacterial systems22. On the other hand, Neutron Spin Echo spectroscopy (NSE) has been shown to be an invaluable tool to explore the dynamics of biomolecules on larger spatial scales, of the order of nanometer, for times up to hundreds of nanoseconds2324252627. NSE has been successfully applied to systems that exhibit long-range signaling modes via domain displacement, as in the case of the NHERF125, Taq polymerase23, and Phosphoglycerate Kinase26, as well as to Alcohol Dehydrogenase, a more compact multimeric protein24.…”