Compensatory and Long-Range Changes in Picosecond–Nanosecond Main-Chain Dynamics upon Complex Formation: 15N Relaxation Analysis of the Free and Bound States of the Ubiquitin-like Domain of Human Plexin-B1 and the Small GTPase Rac1

Abstract

The formation of a complex between Rac1 and the cytoplasmic domain of plexin-B1 is one of the first documented cases of a direct interaction between a small guanosine 5′-triphosphatase (GTPase) and a transmembrane receptor. Structural studies have begun to elucidate the role of this interaction for the signal transduction mechanism of plexins. Mapping of the Rac1 GTPase surface that contacts the Rho GTPase binding domain of plexin-B1 by solution NMR spectroscopy confirms the plexin domain as a GTPase effector protein. Regions neighboring the GTPase switch I and II regions are also involved in the interaction and there is considerable interest to examine the changes in protein dynamics that take place upon complex formation. Here we present main-chain nitrogen-15 relaxation measurements for the unbound proteins as well as for the Rho GTPase binding domain and Rac1 proteins in their complexed state. Derived order parameters, S 2 , show that considerable motions are maintained in the bound state of plexin. In fact, some of the changes in S 2 on binding appear compensatory, exhibiting decreased as well as increased dynamics. Fluctuations in Rac1, already a largely rigid protein on the picosecond-nanosecond timescale, are overall diminished, but isomerization dynamics in the switch I and II regions of the GTPase are retained in the complex and appear to be propagated to the bound plexin domain. Remarkably, fluctuations in the GTPase are attenuated at sites, including helices α6 (the Rho-specific insert helix), α7 and α8, that are spatially distant from the interaction region with plexin. This effect of binding on long-range dynamics appears to be communicated by hinge sites and by subtle conformational changes in the protein. Similar to recent studies on other systems, we suggest that dynamical protein features are affected by allosteric mechanisms. Altered protein fluctuations are likely to prime the Rho GTPase-plexin complex for interactions with additional binding partners.

Structure and Function of the Intracellular Region of the Plexin-B1 Transmembrane Receptor

“…as an isolated dimer (21) and reported here as a dimer bound to Rnd1 GTPase) and in the context of the surrounding GAP domain. Comparison of these structures shows that the conformational differences are very limited and occur only in loops known to be flexible in solution (33). There is no indication from these structures that GTPase binding could disrupt RBD-GAP interactions either directly or indirectly through destabilization of contacts involving the coupling loop.…”

“…5c). They are also known to be flexible in solution (33). Other regions of the RBD structure show remarkably little structural variation, suggesting that the contacts with the GAP domain can be accommodated without significant conformational changes in the RBD structure.…”

Classical Rho Proteins: Biochemistry of Molecular Switch Function and Regulation

“…Although the function of the insert helix has not been elucidated yet, it has been reported to be involved in the Rho-dependent activation of ROCK (Zong et al 2001), phospholipase D (Walker and Brown 2002) and mDia (Lammers et al 2008;Rose et al 2005), and in the Rac-dependent activation of p67phox (Joneson and Bar-Sagi 1997;Karnoub et al 2001;Nisimoto et al 1997) and Plexin B1 (Bouguet-Bonnet and Buck 2008).…”

NMR reveals novel mechanisms of protein activity regulation

“…[33][34][35] Numerous examples are currently available highlighting the inextricable link between protein dynamics and allostery 8,36 . Long-range allosteric coupling between sites through changes in internal dynamics were seen between the nucleotide-binding cleft and the preprotein-binding site in SecA ATPase, 37 between the coordination Na þ site and exosite I in thrombin that regulates enzyme's specificity, 38 between the substrate-binding site and distal loop in dihydrofolate reductase, 39 between the mixed lineage leukemia)-and c-Myb binding sites of the KIX domain of CREB-binding protein, 40 in PDZ signaling domains, 41 the serine protease inhibitor eglin c, 42 upon binding of barstar to the RNase barnase, 43 in the interaction between the Rho GTPasebinding domain and Rac1, 44 upon cyclic nucleotide binding to the exchange protein activated by cAMP, 45,46 a in V-type allosteric enzyme, 47 and in protein kinase A, 48,49 only to mention few recent examples from a long list of systems characterized over the years. Dynamic changes in these systems were accompanied by varying extent of structural changes, ranging from minimal to substantial.…”