Shiou Ru Tzeng

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.

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.

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.

Structure of the ubiquitin‐binding zinc finger domain of human DNA Y‐polymerase η

The ubiquitin‐binding zinc finger (UBZ) domain of human DNA Y‐family polymerase (pol) η is important in the recruitment of the polymerase to the stalled replication machinery in translesion synthesis. Here, we report the solution structure of the pol η UBZ domain and its interaction with ubiquitin. We show that the UBZ domain adopts a classical C2H2 zinc‐finger structure characterized by a ββα fold. Nuclear magnetic resonance titration maps the binding interfaces between UBZ and ubiquitin to the α‐helix of the UBZ domain and the canonical hydrophobic surface of ubiquitin defined by residues L8, I44 and V70. Although the UBZ domain binds ubiquitin through a single α‐helix, in a manner similar to the inverted ubiquitin‐interacting motif, its structure is distinct from previously characterized ubiquitin‐binding domains. The pol η UBZ domain represents a novel member of the C2H2 zinc finger family that interacts with ubiquitin to regulate translesion synthesis.

Allosteric inhibition through suppression of transient conformational states

The ability to inhibit binding or enzymatic activity is key to preventing aberrant behaviors of proteins. Allosteric inhibition is desirable as it offers several advantages over competitive inhibition, but the mechanisms of action remain poorly understood in most cases. Here we show that allosteric inhibition can be effected by destabilizing a low-populated conformational state that serves as an on-pathway intermediate for ligand binding, without altering the proteins ground-state structure. As standard structural approaches are typically concerned with changes in the ground-state structure of proteins, the presence of such a mechanism can go easily undetected. Our data strongly argue for the routine use of NMR tools suited to detect and characterize transiently formed conformational states in allosteric systems. Structure information on such important intermediates can ultimately result in more efficient design of allosteric inhibitors.

Structural basis for regulation of the Crk signaling protein by a proline switch

Proline switches, controlled by cis–trans isomerization, have emerged as a particularly effective regulatory mechanism in a wide range of biological processes. Here we report the structures of both the cis and trans conformers of a proline switch in Crk signaling protein. Proline isomerization toggles Crk between two conformations: an autoinhibitory, stabilized by the intramolecular association of two tandem SH3 domains in the cis form, and an uninhibited, activated conformation promoted by the trans form. In addition to acting as a structural switch the heterogeneous proline recruits cyclophilin A, which accelerates the interconversion rate between the isomers thereby regulating the kinetics of Crk activation. The data provide atomic insight into the mechanisms that underpin the functionality of this binary switch and elucidate its remarkable efficiency. The results also reveal novel SH3 binding surfaces highlighting the binding versatility and expanding the non-canonical ligand repertoire of this important signaling domain.

Solution Structure of a Novel Tryptophan-Rich Peptide with Bidirectional Antimicrobial Activity

Trp-rich antimicrobial peptides play important roles in the host innate defense mechanisms of many plants, insects, and mammals. A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH(2), designated Pac-525, was found to possess improved activity against both gram-positive and -negative bacteria. We have determined that the solution structures of Pac-525 bound to membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The SDS micelle-bound structure of Pac-525 adopts an alpha-helical segment at residues Trp2, Arg3, and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH(2), designated Pac-525(rev), also demonstrates similar antimicrobial activity and structure in membrane-mimetic micelles and vesicles. A variety of biophysical and biochemical methods, including circular dichroism, fluorescence spectroscopy, and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles, suggesting that the antimicrobial activity of Pac-525 may be due to interactions with bacterial membranes.

NMR studies of large protein systems.

Over the recent years, there has been increased interest in applying NMR spectroscopy for the characterization of proteins and protein complexes of large molecular weight. The combination of multidimensional NMR, novel pulse sequences allowing for the selection of slowly relaxing coherence pathways, and the development of a range of labeling techniques has enabled high-resolution NMR analyses of supramolecular systems of even megadalton size. Here, we describe how NMR can be used to obtain structural information in large systems by using as an example the recent structure determination of SecA ATPase (204 kDa) in complex with a signal peptide.

The role of slow and fast protein motions in allosteric interactions

Allostery is fundamentally thermodynamic in nature. Long-range communication in proteins may be mediated not only by changes in the mean conformation with enthalpic contribution but also by changes in dynamic fluctuations with entropic contribution. The important role of protein motions in mediating allosteric interactions has been established by NMR spectroscopy. By using CAP as a model system, we have shown how changes in protein structure and internal dynamics can allosterically regulate protein function and activity. The results indicate that changes in conformational entropy can give rise to binding enhancement, binding inhibition, or have no effect in the expected affinity, depending on the magnitude and sign of enthalpy–entropy compensation. Moreover, allosteric interactions can be regulated by the modulation a low-populated conformation states that serve as on-pathway intermediates for ligand binding. Taken together, the interplay between fast internal motions, which are intimately related to conformational entropy, and slow internal motions, which are related to poorly populated conformational states, can regulate protein activity in a way that cannot be predicted on the basis of the protein’s ground-state structure.