Binding Leverage as a Molecular Basis for Allosteric Regulation

Citation: Mitternacht S, Berezovsky IN ( Binding Leverage as a Molecular Basis for Allosteric Regulation Simon Mitternacht 0 Igor N. Berezovsky 0 Thomas Lengauer, Max-Planck-Institut f ur Informatik, Germany 0 1 Computational Biology Unit/UNI Research, University of Bergen , Bergen , Norway , 2 Department of Informatics, University of Bergen , Bergen , Norway Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design. - Protein function depends on the balance between different conformational states. This balance can be shifted by many external factors that regulate protein activity, including localized perturbations such as ligand binding or phosphorylation. When the perturbation site is not directly adjacent to the site of altered activity the regulation is called allosteric. A classic example of allosteric regulation is the cooperative ligand binding of many oligomeric proteins, where binding of substrate to one subunit affects the ligand affinity in other identical subunits. The early phenomenological MWC (Monod-Wyman-Changeux) [1] and KNF (Koshland-Nemethy-Filmer) [2] models were devised to explain this cooperativity; the first model states that binding stabilizes one of several available states with emphasis on symmetry conservation [3], whereas the latter assumes an induced-fit scenario. Weber showed that both models can be integrated in a general physical framework [4]. Free energy landscape-based descriptions of allostery have introduced the related terms population shift and conformational selection [5,6]. In a recent review Cui and Karplus gave a clear discussion of the relation between the classical models [1,2] and the new views of allostery [5,6], pointing out that the MWC/Weber formalism already includes the idea of population shift [7]. The microscopic mechanisms involved in allostery have been studied at different levels of coarse-graining. Analysis of the effect of different types of perturbations has shown some promise in identifying allosteric sites [8,9]. Ferreiro et al. showed that frustration localized to a few residues facilitates transitions between alternative conformations [10]. Normal modes have been used to quantitatively analyze different energetic and entropic contributions to allostery [11,12,13], and also the major components of conformational change [14,15]. The interaction networks used in normal mode analysis define subunits of coherent dynamics and can be used to identify key residues that maintain this coherence [16,17]. The network description has also been extended to study transmission of allosteric signals throughout the protein [16,18,19, 20,21]. Caution must however be taken against overly mechanistic interpretations of the networks: allosteric regulation is primarily a thermodynamic process. An integral part of the modern understanding of allostery is that the states subject to regulation are part of the intrinsic protein dynamics [3,6,22,23], which to some extent is a truism since states not sampled by the native protein would require infinite binding energies to be given finite Boltzmann weights upon binding. A reasonable interpretation of this concept is however that regulation does not require crossing of large barriers: the relevant states are easily reached from the native basin and are occasionally visited also in the absence of effectors. For example, the allosteric conformational transitions are often well described by low frequency normal modes [14,15]. The existence of purely entropic allosteric proteins [24], where regulation only alters the magnitude of fluctuations around the native state, also shows the importance of intrinsic dynamics. Studies of artificial allosteric inhibitors show that allosteric proteins are often amenable to additional regulation, and that artificial inhibitors stabilize a naturally occurring conformation [25]. These observations give hope for identifying allosteric sites based on intrinsic protein dynamics without doing full scale simulations: it seems that knowledge of basic degrees of freedom, such as low frequency normal modes, or some alternative Allosteric protein regulation is the mechanism by which binding of a molecule to one site in a protein affects the activity at another site. Although the two classical phenomenological models, Monod-Wyman-Changeux (MWC) and Koshland-Ne methy-Filmer (KNF), span from the case of hemoglobin to membrane receptors, they do not describe the intramolecular interactions involved. The coupling between two allosterically connected sites commonly takes place through coherent collective motion involving the whole protein. We therefore introduce a quantity called binding leverage to measure the strength of the coupling between particular binding sites and such motions. We show that high binding leverage is a characteristic of both allosteric sites and catalytic sites, emphasizing that both enzymatic function and allosteric regulation require a coupling between ligand binding and protein dynamics. We also consider the first known case of purely entropic allostery, where ligand binding only affects the amplitudes of fluctuations. We find that the binding site in this protein does not primarily connect to collective motions instead the modulation of fluctuations is controlled from a deeply buried and highly connected site. Finally, sites with high binding leverage but no known biological function could be latent allosteric sites, and thus drug targets. conformations from different crystal structures, gives useful information for finding plausible mechanisms for allosteric regulation. Our goal is to build a general molecular description of allosteric regulation that allows prediction of biological and latent allosteric sites, as well as catalytic sites, from crystal structures. We restrict ou (...truncated)