Charalampos G. Kalodimos

Dynamically driven protein allostery

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.

Structural Basis for Signal-Sequence Recognition by the Translocase Motor SecA as Determined by NMR

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.

Protein activity regulation by conformational entropy

How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data. It is likely that changes in the internal motions of proteins have a major role in regulating protein activity, but the nature of their contributions remains elusive, especially in quantitative terms. Here we show that changes in conformational entropy can determine whether protein–ligand interactions will occur, even among protein complexes with identical binding interfaces. We have used NMR spectroscopy to determine the changes in structure and internal dynamics that are elicited by the binding of DNA to several variants of the catabolite activator protein (CAP) that differentially populate the inactive and active DNA-binding domain states. We found that the CAP variants have markedly different affinities for DNA, despite the CAP−DNA-binding interfaces being essentially identical in the various complexes. Combined with thermodynamic data, the results show that conformational entropy changes can inhibit the binding of CAP variants that are structurally poised for optimal DNA binding or can stimulate the binding activity of CAP variants that only transiently populate the DNA-binding-domain active state. Collectively, the data show how changes in fast internal dynamics (conformational entropy) and slow internal dynamics (energetically excited conformational states) can regulate binding activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.

Dynamic activation of an allosteric regulatory protein

Allosteric regulation is used as a very efficient mechanism to control protein activity in most biological processes, including signal transduction, metabolism, catalysis and gene regulation. Allosteric proteins can exist in several conformational states with distinct binding or enzymatic activity. Effectors are considered to function in a purely structural manner by selectively stabilizing a specific conformational state, thereby regulating protein activity. Here we show that allosteric proteins can be regulated predominantly by changes in their structural dynamics. We have used NMR spectroscopy and isothermal titration calorimetry to characterize cyclic AMP (cAMP) binding to the catabolite activator protein (CAP), a transcriptional activator that has been a prototype for understanding effector-mediated allosteric control of protein activity. cAMP switches CAP from the ‘off’ state (inactive), which binds DNA weakly and non-specifically, to the ‘on’ state (active), which binds DNA strongly and specifically. In contrast, cAMP binding to a single CAP mutant, CAP-S62F, fails to elicit the active conformation; yet, cAMP binding to CAP-S62F strongly activates the protein for DNA binding. NMR and thermodynamic analyses show that despite the fact that CAP-S62F-cAMP2 adopts the inactive conformation, its strong binding to DNA is driven by a large conformational entropy originating in enhanced protein motions induced by DNA binding. The results provide strong evidence that changes in protein motions may activate allosteric proteins that are otherwise structurally inactive.

Computational design of ligand-binding proteins with high affinity and selectivity.

The ability to design proteins with high affinity and selectivity for any given small molecule is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition. Attempts to rationally design ligand-binding proteins have met with little success, however, and the computational design of protein–small-molecule interfaces remains an unsolved problem. Current approaches for designing ligand-binding proteins for medical and biotechnological uses rely on raising antibodies against a target antigen in immunized animals and/or performing laboratory-directed evolution of proteins with an existing low affinity for the desired ligand, neither of which allows complete control over the interactions involved in binding. Here we describe a general computational method for designing pre-organized and shape complementary small-molecule-binding sites, and use it to generate protein binders to the steroid digoxigenin (DIG). Of seventeen experimentally characterized designs, two bind DIG; the model of the higher affinity binder has the most energetically favourable and pre-organized interface in the design set. A comprehensive binding-fitness landscape of this design, generated by library selections and deep sequencing, was used to optimize its binding affinity to a picomolar level, and X-ray co-crystal structures of two variants show atomic-level agreement with the corresponding computational models. The optimized binder is selective for DIG over the related steroids digitoxigenin, progesterone and β-oestradiol, and this steroid binding preference can be reprogrammed by manipulation of explicitly designed hydrogen-bonding interactions. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics and diagnostics.

Protein dynamics and allostery: an NMR view

Allostery, the process by which distant sites within a protein system are energetically coupled, is an efficient and ubiquitous mechanism for activity regulation. A purely mechanical view of allostery invoking only structural changes has developed over the decades as the classical view of the phenomenon. However, a fast growing list of examples illustrate the intimate link between internal motions over a wide range of time scales and function in protein-ligand interactions. Proteins respond to perturbations by redistributing their motions and they use fluctuating conformational states for binding and conformational entropy as a carrier of allosteric energy to modulate association with ligands. In several cases allosteric interactions proceed with minimal or no structural changes. We discuss emerging paradigms for the central role of protein dynamics in allostery.

Crk and CrkL adaptor proteins: networks for physiological and pathological signaling

The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses.

Structural basis for cAMP-mediated allosteric control of the catabolite activator protein

The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by ≈60°, and translation, by ≈7 Å, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.

Structural Basis for Protein Antiaggregation Activity of the Trigger Factor Chaperone

Introduction Molecular chaperones prevent aggregation and misfolding of proteins in the cellular environment and are thus central to maintaining protein homeostasis. Molecular chaperones are thought to recognize and bind to exposed hydrophobic regions of the unfolded proteins, thereby shielding these regions from the solvent. If unprotected, the proteins would likely aggregate or misfold to bury the hydrophobic residues. Despite the central importance of the binding of chaperones to unfolded proteins, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded proteins is primarily due to technical challenges originating in the size and dynamic nature of these complexes. Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein.Structural basis of PhoA binding by TF. PhoA (blue/gray) is captured in an unfolded state by three TF chaperone molecules (orange). Complex formation is mediated by multivalent binding of hydrophobic surfaces, which are shielded from water, thereby preventing folding and, at the same time, aggregation of the substrate protein. Rationale Recent advances in nuclear magnetic resonance (NMR) and isotope labeling approaches make it possible to study large, dynamic complexes. We used NMR spectroscopy to characterize the binding of the 48-kD unfolded alkaline phosphatase (PhoA) to the 50-kD trigger factor (TF) chaperone. We obtained atomic insight into the dynamic binding and determined the solution structure of PhoA captured in an extended, unfolded state by three TF molecules. Based on our NMR studies, we gained insight into how TF rescues an aggregation-prone protein and how it exerts its unfoldase activity. Results We show that TF uses multiple sites, which are located in two different domains and extend over a distance of ~90 Å, to bind to several regions of the unfolded PhoA that are dispersed throughout its entire length. Three TF molecules are required to interact with the entire length of PhoA, giving rise to a ~200-kD complex in solution. The TF-PhoA interactions are mediated primarily by hydrophobic contacts. TF interacts with PhoA in a highly dynamic fashion, giving rise to a rugged landscape for the free energy of interaction. As the number and length of the PhoA regions engaged by TF increases, a more stable complex gradually emerges. The multivalent binding keeps PhoA in an extended, unfolded conformation. Crucially, even the lowest-energy TF-PhoA complex remains rather dynamic with a lifetime of ~20 ms. The structural data of the three TF molecules in complex with different regions of PhoA reveal how the same binding sites within a molecular chaperone can recognize and interact with a large number of substrates with unrelated primary sequences. This promiscuous recognition is further enabled by the notable plasticity of the substrate-binding sites in TF. We finally show that TF in the cytosol prevents aggregation by interacting transiently with the low-populated, aggregation-prone unfolded state of the substrate but acts as a powerful unfoldase when it is bound at the ribosome and thus is colocalized with translating substrate. Conclusion The structural data reveal a multivalent binding mechanism between the chaperone and its protein substrate. This mechanism of binding presents several advantages as it enables chaperones to function as holdases and unfoldases by exerting forces to retain proteins in the unfolded state and at the same time protect them from aggregation by shielding their exposed hydrophobic regions. Given the existence of multiple binding sites in other molecular chaperones, this may present a general mechanism for the action of molecular chaperones. The fast kinetics of substrate binding enables chaperones to interact with transiently exposed, aggregation-prone regions of unstable proteins in the cytosol, thereby preventing their aggregation and increasing their solubility. Nuclear magnetic resonance data show how molecular chaperones recognize and prevent aggregation and misfolding of unfolded proteins. [Also see Perspective by Gamerdinger and Deuerling] Molecular chaperones prevent aggregation and misfolding of proteins, but scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms. We report the solution structure, dynamics, and energetics of three trigger factor (TF) chaperone molecules in complex with alkaline phosphatase (PhoA) captured in the unfolded state. Our data show that TF uses multiple sites to bind to several regions of the PhoA substrate protein primarily through hydrophobic contacts. Nuclear magnetic resonance (NMR) relaxation experiments show that TF interacts with PhoA in a highly dynamic fashion, but as the number and length of the PhoA regions engaged by TF increase, a more stable complex gradually emerges. Multivalent binding keeps the substrate protein in an extended, unfolded conformation. The results show how molecular chaperones recognize unfolded polypeptides and, by acting as unfoldases and holdases, prevent the aggregation and premature (mis)folding of unfolded proteins. Recognize and Protect Molecular chaperones play a key role in maintaining protein homeostasis in the cell by preventing protein aggregation and misfolding. Chaperone-substrate complexes tend to be large and dynamic, making structure determination challenging. Saio et al. (10.1126/science.1250494; see the Perspective by Gamerdinger and Deuerling) used advanced NMR spectroscopy techniques to determine the structure of three trigger factor (TF) chaperone molecules in complex with the unfolded substrate, alkaline phosphatase (PhoA), and of each of the TFs in complex with the relevant region of PhoA. TF binds at multiple sites on PhoA through hydrophobic contacts, thus shielding these residues from solvent and preventing aggregation. The stability of the complex increases as longer PhoA regions are engaged by TF, and the multivalent binding keeps the substrate in an extended conformation.

Signal peptides are allosteric activators of the protein translocase

Extra-cytoplasmic polypeptides are usually synthesized as ‘preproteins’ carrying amino-terminal, cleavable signal peptides and secreted across membranes by translocases. The main bacterial translocase comprises the SecYEG protein-conducting channel and the peripheral ATPase motor SecA. Most proteins destined for the periplasm and beyond are exported post-translationally by SecA. Preprotein targeting to SecA is thought to involve signal peptides and chaperones like SecB. Here we show that signal peptides have a new role beyond targeting: they are essential allosteric activators of the translocase. On docking on their binding groove on SecA, signal peptides act in trans to drive three successive states: first, ‘triggering’ that drives the translocase to a lower activation energy state; second, ‘trapping’ that engages non-native preprotein mature domains docked with high affinity on the secretion apparatus; and third, ‘secretion’ during which trapped mature domains undergo several turnovers of translocation in segments. A significant contribution by mature domains renders signal peptides less critical in bacterial secretory protein targeting than currently assumed. Rather, it is their function as allosteric activators of the translocase that renders signal peptides essential for protein secretion. A role for signal peptides and targeting sequences as allosteric activators may be universal in protein translocases.