Individual metal ligands play distinct functional roles in the zinc sensor Staphylococcus aureus CzrA

Recent studies on metalloregulatory proteins suggest that coordination number/geometry and metal ion availability in a host cytosol are key determinants for biological specificity. Here, we investigate the contribution that individual metal ligands of the alpha5 sensing site of Staphylococcus aureus CzrA (Asp84, His86, His97', and His100') make to in vitro metal ion binding affinity, coordination geometry, and allosteric negative regulation of DNA operator/promoter region binding. All ligand substitution mutants exhibit significantly reduced metal ion binding affinity (K(Me)) by > or =10(3) M(-1). Substitutions of Asp84 and His97 give rise to non-native coordination geometries upon metal binding and are non-functional in allosteric coupling of metal and DNA binding (DeltaG(coupling) approximately 0 kcal mol(-1)). In contrast, His86 and His100 could be readily substituted with potentially liganding (Asp, Glu) and poorly liganding (Asn, Gln) residues with significant native-like tetrahedral metal coordination geometry retained in these mutants, leading to strong functional coupling (DeltaG(coupling) > or = +3.0 kcal mol(-1)). 1H-(15)N heteronuclear single quantum coherence (HSQC) spectra of wild-type and mutant CzrAs reveal that all H86 and H100 substitution mutants undergo 4 degrees structural switching on binding Zn(II), while D84N, H97N and H97D CzrAs do not. Thus, only those variant CzrAs that retain some tetrahedral coordination geometry characteristic of wild-type CzrA upon metal binding are capable of driving 4 degrees structural conformational changes linked to allosteric regulation of DNA binding in vitro, irrespective of the magnitude of K(Me).     

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.     

Arm-domain interactions can provide high binding cooperativity

Peptidyl arms extending from one protein domain to another protein domain mediate many important interactions in biology. A well-studied example of this type of protein-protein interaction occurs between the yeast homeodomain proteins, MAT alpha2 and MAT a1, which form a high-affinity heterodimer on DNA. The carboxyl-terminal arm extending from MAT alpha2 to MAT a1 has been proposed to produce an allosteric conformational change in the a1 protein that generates a very large increase in the DNA binding affinity of a1. Although early studies lent some support to this model, a more recent crystal structure determination of the free a1 protein argues against any allosteric change. This note presents a thermodynamic argument that accounts for the proteins' binding behavior, so that allosteric conformational changes are not required to explain the large affinity increase. The analysis presented here should be useful in analyzing binding behavior in other systems involving arm interactions.     

Binding leverage as a molecular basis for allosteric regulation

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.     

Computation of Conformational Coupling in Allosteric Proteins

In allosteric regulation, an effector molecule binding a protein at one site induces conformational changes, which alter structure and function at a distant active site. Two key challenges in the computational modeling of allostery are the prediction of the structure of one allosteric state starting from the structure of the other, and elucidating the mechanisms underlying the conformational coupling of the effector and active sites. Here we approach these two challenges using the Rosetta high-resolution structure prediction methodology. We find that the method can recapitulate the relaxation of effector-bound forms of single domain allosteric proteins into the corresponding ligand-free states, particularly when sampling is focused on regions known to change conformation most significantly. Analysis of the coupling between contacting pairs of residues in large ensembles of conformations spread throughout the landscape between and around the two allosteric states suggests that the transitions are built up from blocks of tightly coupled interacting sets of residues that are more loosely coupled to one another.     

Long-range interactions between DNA-bound ligands

We have studied the interaction of the A:T specific minor-groove binding ligand 4′, 6-diamidino-2-phenylindole (DAPI) with synthetic DNA oligomers containing specific binding sites in order to investigate possible long-range interactions between bound ligands. We find that DAPI binds cooperatively to the oligomers. The degree of cooperativity increases with increasing number of binding sites and decreases with the separation between them. This dependence is paralleled by changes in the induced circular dichroism spectrum of DAPI, which decreases in intensity at 335 nm and increases at 365 nm. These results are consistent with an allosteric interaction of DAPI with DNA, where bound ligands cooperatively alter the structure of the DNA molecule. This structural change seems possible to induce under various conditions, including physiological. One consequence of allosteric binding is that ligands bound at a distance from each other sense each other's presence and influence each other's properties. If some regulatory proteins the same conformational change as DAPI, novel mechanisms for controlling gene expression can be anticipated.     

The changing landscape of protein allostery

It is becoming increasingly clear that the fundamental capacity to undergo conformational change in response to ligand binding is intrinsic to proteins. This property confers on proteins the ability to be allosterically modulated in order to shift substrate binding affinities, alter enzymatic activity or regulate protein-protein interaction. How this allosteric modulation occurs--the pathways of communication, the shifting of conformational ensembles and the altered molecular dynamics--has received considerable attention during the past two years. Recent progress has helped outline the molecular origins of allostery in proteins as diverse as Hsp70 molecular chaperones and signal integrating proteins, such as WASP. In addition, allosteric properties have been successfully engineered into proteins for drug design or the development of novel biosensors. Methodological advances have provided exciting prospects for new insights and new biological roles of allosteric systems have been uncovered.     

Allosteric regulation and catalysis emerge via a common route

Allosteric regulation of protein function is a mechanism by which an event in one place of a protein structure causes an effect at another site, much like the behavior of a telecommunications network in which a collection of transmitters, receivers and transceivers communicate with each other across long distances. For example, ligand binding or an amino acid mutation at an allosteric site can alter enzymatic activity or binding affinity in a distal region such as the active site or a second binding site. The mechanism of this site-to-site communication is of great interest, especially since allosteric effects must be considered in drug design and protein engineering. In this review, conformational mobility as the common route between allosteric regulation and catalysis is discussed. We summarize recent experimental data and the resulting insights into allostery within proteins, and we discuss the nature of future studies and the new applications that may result from increased understanding of this regulatory mechanism.     

Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor.

Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain.     

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.     

Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function

Protein structural change underlies many signal transduction processes. Although end-state structures are known for various allosteric proteins, intermediates are difficult to observe. Recently, targeted molecular dynamics simulation (TMD) was used to examine the conformational transition and predict relevant intermediates for wild-type lactose repressor (LacI). A catalog of involved residues suggests that the transition of this homodimer is asymmetric and that K84 is a prominent participant in the dynamic N-subdomain interface. Previous experiments indicated that hydrophobic substitutions at position 84 engender slowed, biphasic inducer binding kinetics, which might reflect the same phenomena observed in TMD. Here, we report biochemical confirmation that DNA and inducer binding remain allosterically linked in K84A and K84L, albeit with a differential smaller than that found in wild-type LacI. Other features of these mutant proteins are consistent with an allosteric conformational shift that approximates that of the wild type. As a consequence, these repressors can be utilized to explore an unanswered question about LacI function: How many inducers (one or two per dimer) are required to diminish operator affinity? The biphasic natures of the K84L and K84A inducer association rates allow direct correlation between the two distinct inducer binding events and operator release. Indeed, the kinetics of operator release for the K84A and K84L closely parallel those for the second inducer binding event. Together with implications from previous equilibrium results for wild-type and mutant proteins, these kinetic data demonstrate that binding of two inducers per dimeric DNA binding unit is required to release the operator in these variant LacI proteins.