S-(3,4-dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homolog MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We presently show that A22 binds MreB with at least micromolar affinity in its nucleotide binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the timecourse and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents MreB assembly into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

The heterodimeric actin-capping protein (CP) can be inhibited by polyphosphoinositides, which may be important for actin polymerization at membranes in cells. Here, we have identified a conserved set of basic residues on the surface of CP that are important for the interaction with phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Computational docking studies predicted the identity of residues involved in this interaction, and functional and physical assays with site-directed mutants of CP confirmed the prediction. The PIP 2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP 2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP 2 rapidly converted capped actin filaments to a growing state, consistent with uncapping. Together, these results extend our understanding of how CP binds to the barbed end of the actin filament, and they support the idea that CP can "wobble" when bound to the barbed end solely by the C-terminal "tentacle" of its ␤-subunit.

Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca2+-sensitivity of the thin filament have been linked to disease. Tikunova and Davis have engineered a series of cNTnC mutations that altered Ca2+ binding properties and studied the effects on the Ca2+ sensitivity of the thin filament and contraction [Tikunova and Davis (2004) J Biol Chem279, 35341–35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in cNTnC Ca2+ binding affinity and Ca2+ sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca2+ sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca2+ and cTnI to cTnC. NMR chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca2+ sensitivity can do so by modulating Ca2+ and cTnI binding.

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 micros of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Phi values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated.

The protein folding problem is often studied by comparing the mechanisms of proteins sharing the same structure but different sequence. The recent design of the two proteins G A 88 and G B 88, displaying different structures and functions while sharing 88% sequence identity (49 out of 56 amino acids), allows the unique opportunity for a complementary approach. At which stage of its folding pathway does a protein commit to a given topology? Which residues are crucial in directing folding mechanisms to a given structure? By using a combination of biophysical and computational techniques, we have characterized the folding of both G A 88 and G B 88. We show that, contrary to expectation, G B 88, characterized by a native ␣؉␤ fold, displays in the denatured state a content of native-like helical structure greater than G A 88, which is all-␣ in its native state. Both experiments and simulations indicate that such residual structure may be tuned by changing pH. Thus, despite the high sequence identity, the folding pathways for these two proteins appear to diverge as early as in the denatured state. Our results suggest a mechanism whereby protein topology is committed very early along the folding pathway, being imprinted in the residual structure of the denatured state.