Chapter 5 - Proteins MOVE! Protein dynamics and long-range allostery in cell signaling

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Abstract

An emerging point of view in protein chemistry is that proteins are not the static objects that are displayed in textbooks but are instead dynamic actors. Protein dynamics plays a fundamental role in many diseases, and spans a large hierarchy of timescales, from picoseconds to milliseconds or even longer. Nanoscale protein domain motion on length scales comparable to protein dimensions is key to understanding how signals are relayed through multiple protein–protein interactions. A canonical example is how the scaffolding proteins NHERF1 and ezrin work in coordination to assemble crucial membrane complexes. As membrane–cytoskeleton scaffolding proteins, these provide excellent prototypes for understanding how regulatory signals are relayed through protein–protein interactions between the membrane and the cytoskeleton. Here, we review recent progress in understanding the structure and dynamics of the interaction. We describe recent novel applications of neutron spin echo spectroscopy to reveal the dynamic propagation of allosteric signals by nanoscale protein motion, and present a guide to the future study of dynamics and its application to the cure of disease.

Introduction

Proteins move! This simple statement encapsulates a wide variety of phenomena that are central for the understanding of life and for the cure of disease. Proteins are composed of multiple domains, whose flexibility and mobility lead to a great deal of versatility in their function. Protein dynamics (particularly at the domain level) is a controlling influence in the allosteric formation of protein complexes, in catalysis, in cell signaling and regulation, in metabolic transport, and in cellular locomotion. Yet, despite the importance of protein domain dynamics, the study of this field is in its infancy, largely because of the paucity of biophysical methods that are able to probe this regime. We here provide a review of some of the field, and propose a roadmap for future exploration. In order to understand what is known about protein dynamics and the significance of the challenges ahead, it is essential to review some of the relevant concepts. In order to motivate this discussion, we will first present a review of the biological relevance of long-range allosteric effects that couple the dynamics of protein domains.

In cells, membrane channels and receptors are often assembled into macromolecular complexes in specialized subcellular domains for the dynamic control of diverse cellular events. For instance, forming “quarternary” complexes of receptors, such as the EGF receptor or the PDGF receptor is necessary for initializing cascades of signaling events for cell growth and proliferation (Schlessinger, 1988). The function of ion transport proteins, such as the cystic fibrosis transmembrane conductance regulator (CFTR) or sodium–phosphate cotransporter 2a (NaPiT2a), is regulated by a network of interactions with other membrane proteins, such as the G-protein coupled receptors and other ion channels by forming membrane oligomers either directly, or via cytosolic proteins as adapters or scaffolds. Forming large adherence membrane complexes at the cell–cell junctions is essential to maintain tissue integrity and to suppress tumor cell invasion (Yap et al., 1997, Perez-Moreno et al., 2003, Pujuguet et al., 2003). Understanding how transmembrane protein complexes are regulated and disregulated in disease state can help to identify elements as target to treat various diseases.

The mammalian Na+/H+ exchange regulatory factor (NHERF) family proteins are scaffolding proteins that assemble macromolecular complexes of transmembrane proteins, and regulate receptor signaling and ion transport (Shenolikar et al., 2004, Lamprecht and Seidler, 2006, Weinman et al., 2006). Members of this protein family, which contain two or more copies of modular PDZ (PSD-95/Discs-large/ZO-1) domains (Fig. 1), localize in the apical membrane region of polarized epithelial cells (Donowitz et al., 2005, Thelin et al., 2005). The PDZ domains are protein–protein interaction modules that are capable of binding to specific PDZ-binding motifs residing in the cytoplasmic portion of a large number of transmembrane proteins (Harris and Lim, 2001). Scaffolding proteins containing multiple PDZ domains and/or other protein–protein interaction modular domains can assemble membrane complexes as well as bring the membrane complexes into proximity with other cytosolic signaling molecules to assemble highly regulated signaling complexes (Sheng and Sala, 2001).

As the first member of the NHERF family, NHERF1 consists of two PDZ domains, PDZ1 and PDZ2 that bind to membrane proteins. NHERF1 also contains carboxy-terminal domain that binds to the membrane–cytoskeleton linker protein ezrin (EB) (Reczek and Bretscher, 1998) (Fig. 1). NHERF1 was shown to interact with ezrin, and therefore is also called ezrin-binding protein 50 or EBP50 (Reczek et al., 1997). The PDZ domains of NHERF1 interact with a number of transmembrane proteins. Some of the NHERF target proteins are implicated in human diseases (Takahashi et al., 2006, Kwon et al., 2007, Sizemore et al., 2007). The well-known functions of NHERF1 include assembling signaling complexes and regulating the endocytic recycling of the CFTR, cell surface adhesion and antiadhesion proteins such as podocalyxin, G-protein coupled receptors, and tyrosine kinase receptors PDGFR and EGFR (Hall et al., 1998, Cao et al., 1999, Ko et al., 2004, Schmieder et al., 2004, Weinman et al., 2006). NHERF1 mutations, which affect its ability to assemble the transmembrane NaPiT2a, are correlated with impaired renal phosphate reabsorption in patients with chronicle kidney disease (Karim et al., 2008). Altered subcellular localization of NHERF1 is associated with breast cancer progression (Mangia et al., 2009).

The function of NHERF1 and the interactions of NHERF1 with membrane transport proteins and receptors have been reviewed earlier (Shenolikar et al., 2004, Weinman et al., 2006). Recent progress in understanding the structure and function of ezrin and other ezrin–moesin–radixin (ERM) proteins has been reviewed (Bretscher et al., 2002, Fievet et al., 2007, McClatchey and Fehon, 2009, Fehon et al., 2010). Here, we first summarize several representative cases that identify NHERF1 as an important factor to assemble complexes of transmembrane receptors or transport proteins. We review the findings that the assembling of signaling complexes by NHERF1 is allosterically regulated. These biochemical studies illustrate that scaffolding or adapter proteins not only function as scaffolds to dock signaling partners but are also regulated transistors and switches that control the effective propagation of signals from a remote site to a specific location over a long distance. We then review the studies that aimed at understanding the structural and dynamic mechanisms of NHERF1 and its interactions with the membrane–cytoskeleton linker protein ezrin in the allosteric regulation of the assembly of membrane complexes. We show that the long-range allosteric-binding behavior is communicated through interdomain conformational and dynamic changes (Li et al., 2009, Bhattacharya et al., 2010, Farago et al., 2010). A recent study using a novel neutron spin echo (NSE) spectroscopy reveals the activation of long-range interdomain motions in NHERF1 on nanometer length scales and on submicrosecond timescale upon binding to ezrin (Farago et al., 2010). Protein domain motion on these timescales and on length scales comparable to protein dimensions can thus propagate allosteric binding signals dynamically. The long-range conformational changes and nanoscale dynamics during the interactions of NHERF1 and ezrin provide a paradigm for studying how cellular signals are transmitted allosterically over a long distance in the cellular signaling network.

Section snippets

CFTR

The PDZ domains of NHERF1 interact with the C-terminal tail of CFTR (Hall et al., 1998a). Interaction of NHERF1 with CFTR increases the polarized expression of CFTR in the apical plasma membrane, as well as enhances the vectorial transport of chloride ions (Moyer et al., 2000, Raghuram et al., 2001). Moreover, NHERF1 overexpression increases the cell surface expression of a disease-causing mutant of CFTR with a deletion at amino acid Phe508 (ΔF508) (Guerra et al., 2005, Bossard et al., 2007,

Signal Transduction by Allosteric Scaffolding Protein Interactions

The important feature of NHERF1 is its binding to ezrin and other members of the ERM proteins. The interaction of NHERF1 with F-actin is due to the NHERF1 C-terminal EBD (Reczek and Bretscher, 1998). Ezrin and other ERM proteins are the membrane–cytoskeleton adapter or scaffolding proteins that link cell membrane to the F-actin cytoskeleton. The ERM proteins contain an N-terminal FERM (4.1 ezrin–radixin–moesin) domain of about 300 amino acid residues, and a C-terminal actin-binding domain that

Structural Basis of Autoinhibition and Long-Range Allostery in NHERF1

The above examples show that the assembly of membrane complexes by NHERF1 is allosterically regulated. The allosteric regulation is not confined to a single protein, but rather it is an allosteric relay of signals through a chain of multiple protein–protein interactions. Such a relay of signals, in a Rube Goldberg device style, can also be found in almost all other signaling pathways (Ma and Nussinov, 2009, Scott and Pawson, 2009). The relay of signals, using multiple proteins as transistors,

Dynamic Propagation of Allosteric Signals by Nanoscale Protein Motion

The above examples demonstrate that allostery of signals are transmitted over a long distance within a single protein, as well as in multiple protein–protein interactions. There is increasing evidence that the transmission of allosteric-binding signals requires both conformational changes and protein motion (Kern and Zuiderweg, 2003). Protein dynamics can initiate and control protein function. Protein motion regulates the transition state dynamics of enzyme catalysis (Benkovic and

Summary and Perspective

Allosteric transduction of cellular signals by multiple protein–protein interactions has emerged as an important theme to elucidate the mechanisms of hierarchy signaling pathways and networks. Nanoscale protein domain motions on length scales comparable to protein dimensions hold the key to understanding how signals are relayed through multiple protein–protein interactions. We have explained in this review our view of the present state of protein dynamics, and described our view of the future

Acknowledgment

This work is supported by National Institutes of Health Grants R01 HL086496.

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