Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Blavatnik Awards for Young Scientists Protein function and allostery: a dynamic relationship Charalampos G. Kalodimos Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey Address for correspondence: Charalampos G. Kalodimos, Department of Chemistry and Chemical Biology, Rutgers University, 599 Taylor Rd., Piscataway, NJ 08854. babis@rutgers.edu Allostery is a fundamental process by which distant sites within a protein system sense each other. Allosteric regulation is such an efficient mechanism that it is used to control protein activity in most biological processes, including signal transduction, metabolism, catalysis, and gene regulation. Over recent years, our view and understanding of the fundamental principles underlying allostery have been enriched and often utterly reshaped. This has been especially so for powerful techniques such as nuclear magnetic resonance spectroscopy, which offers an atomic view of the intrinsic motions of proteins. Here, I discuss recent results on the catabolite activator protein (CAP) that have drastically revised our view about how allosteric interactions are modulated. CAP has provided the first experimentally identified system showing that (i) allostery can be mediated through changes in protein motions, in the absence of changes in the mean structure of the protein, and (ii) favorable changes in protein motions may activate allosteric proteins that are otherwise structurally inactive. Keywords: protein allostery protein dynamics NMR spectroscopy Introduction A fundamental question in allostery is how per- turbation at one site is transmitted through the protein to remote sites to effect binding or enzy- matic activity regulation.1���5 It is generally thought that changes in protein shape and bonding interac- tions, which are considered to contribute primarily to enthalpy, are necessary to propagate binding sig- nals to remote sites.1 This purely mechanical view of allostery���invoking only structural changes��� was advanced and established as the classical view of the phenomenon by the early crystallographic work on allosteric systems, such as hemoglobin and several enzymes.1 However, because allostery is fundamentally thermodynamic in nature, long- range communication may be mediated not only by changes in the mean conformation (enthalpic contribution), but also by changes in the dynamic fluctuations about the mean conformation (en- tropic contribution).6 Indeed, the possibility of allosteric regulation through dynamic (entropic) mechanisms has long been recognized,7 at least at the theoretical level, but has been difficult to prove experimentally.8,9 Nuclear magnetic resonance (NMR) spec- troscopy is one of the most powerful tools for the characterization of biomolecular systems. A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynam- ics of biomolecules.10 In addition, NMR can provide site-resolved information about the conformation entropy of binding,11���13 and about energetically ex- cited conformational states.14,15 NMR characteriza- tion of CAP has recently provided an unprecedented insightintotheintimatelinkbetweenproteinintrin- sic motions and function.16���18 CAP holds an esteemed role in biochemistry history.19 It has been described in countless text- books as a canonical example of effector-mediated allosteric regulation as well as a prototypic activa- tor of transcription initiation.20 CAP is a 50 kDa homodimer with each subunit organized in two distinct domains: (i) an N-terminal cAMP-binding domain (CBD) (residues 1���136), which contains the cyclic nucleotide���binding module and a long - helix (C-helix) that mediates dimerization through formation of an intersubunit coiled coil, and (ii) a C-terminal DNA-binding domain (DBD) (residues 139���209), which contains a helix-turn-helix (HTH) doi: 10.1111/j.1749-6632.2011.06319.x Ann. N.Y. Acad. Sci. xxxx (2012) 1���6 c 2012 New York Academy of Sciences. 1

Protein function and allostery Kalodimos Figure 1. Effect of the sequential, anticooperative binding of cAMP to CBD of CAP (CAPN). Binding of the first cAMP changes the structure of the liganded subunit but has no effect on the mean structure of the unliganded subunit. In contrast, the slow motions ( s-ms) of the unliganded subunit are enhanced (thicker red line). Binding of the second cAMP suppresses the fast motions (ps-ns) on both subunits (blue color). As a result, binding of the second cAMP incurs an unfavorable conformational entropy change, which reduces the affinity of the second cAMP. Thus, the negative cooperativity is entirely of an entropic nature (from Ref. 16). motif for binding to DNA.21 The two domains are linked by a hinge region (residues 137���138). cAMP elicits an allosteric transition that switches CAP from the ������off������ state, which binds DNA weakly and nonspecifically, to the ���on��� state, which binds DNA strongly and specifically.17,22 Dynamically driven protein allostery Two cAMP molecules bind to dimeric CAP with negative cooperativity.16 We exploited the strong negative cooperativity of cAMP binding to CBD of CAP to ���freeze��� binding conformations at in- termediate stages.16 The intermediate stages are the key conformational states, as they ���contain��� the in- formation about how the allosteric sites commu- nicate.23,24 These intermediate states are typically difficult to stabilize and characterize. Based on chemical shift perturbation, which is a very sensitive measure of changes in the average protein confor- mation, we found that binding of the first cAMP to CAP did not induce long-range structural effects to the unliganded subunit (Fig. 1). Thus, the mean conformation of the unfilled cAMP site at the unlig- anded subunit of the intermediate-state complex is not at all affected by the presence of the first cAMP, suggesting that the contribution of the ���structural��� component to the observed negative cooperativity is negligible. To understand how protein dynamics adjust along the allosteric reaction coordinate, the back- bone motions of CAP were measured as a function of the cAMP ligation state over a wide range of functionally relevant timescales by measuring re- laxation rates by NMR. Slow domain motions on the micro- to millisecond ( s���ms) scale are bio- logically very important because they are close to the timescales on which functional processes take place, and they indicate the presence of energeti- cally excited conformational states.25���27 Motions on the pico- to nanosecond (ps���ns) fast timescale are also important because of their strong effect on the entropy of the system.28,29 In contrast to the absence of structural changes, the intrinsic motions of CAP residues in the unliganded subunit were strongly af- fected upon cAMP binding (Fig. 1). Thus, it appears that the unliganded subunit ���senses��� the presence of the ligand (cAMP) in the liganded subunit only throughchangesinproteinmotionsbutnotthrough structural changes. Notably, the data indicated stim- ulation of fluctuations about the mean structure on the slow ( s���ms) timescale, despite the fact that no change in the mean structure was detected. It is par- ticularly noteworthy that slow and fast motions of residues located at distant regions were affected in the absence of a visible connectivity pathway. This result further undermines the mechanical view of allostery, wherein binding effects are assumed to propagate through a series of conformational dis- tortions. Rather, the ligand-induced redistribution of the protein���s dynamic fluctuations affects regions linked by cooperative interactions, thereby provid- ingameansofpropagatingtheallostericsignaltothe distal site even in the absence of structural changes. Interestingly, the thermodynamic basis of the ob- served anti-cooperative binding of cAMP to CAP is entirely of entropic nature. This finding sug- gestedthattheobservedextensivechangesinprotein motions upon sequential cAMP binding were the most probable source of the large difference in en- tropy change between the two cAMP binding steps. 2 Ann. N.Y. Acad. Sci. xxxx (2012) 1���6 c 2012 New York Academy of Sciences.