Michelle E. McCully

A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.

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 homologue 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 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 time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB 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.

Structure/Function Analysis of the Interaction of Phosphatidylinositol 4,5-Bisphosphate with Actin-capping Protein IMPLICATIONS FOR HOW CAPPING PROTEIN BINDS THE ACTIN FILAMENT

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 (PIP2). 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 PIP2 binding site overlaps with the more important of the two known actin-binding sites of CP. Correspondingly, we observed that loss of PIP2 binding correlated with loss of actin binding among the mutants. Using TIRF (total internal reflection fluorescence) microscopy, we observed that PIP2 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 β-subunit.

Microscopic Reversibility of Protein Folding in Molecular Dynamics Simulations of the Engrailed Homeodomain

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 Denatured State Dictates the Topology of Two Proteins with Almost Identical Sequence but Different Native Structure and Function

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 GA88 and GB88, 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 GA88 and GB88. We show that, contrary to expectation, GB88, characterized by a native α+β fold, displays in the denatured state a content of native-like helical structure greater than GA88, which is all-α 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.

Refolding the Engrailed Homeodomain: Structural Basis for the Accumulation of a Folding Intermediate

The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in approximately 15 mus. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to why rearrangements of intermediates occur slowly. To address this problem, we performed 46 all-atom molecular dynamics refolding simulations in explicit water, for a total of 15 micros of simulation time. The simulations started from an intermediate state structure that was generated in an unfolding simulation at 498 K and was then quenched to folding-permissive temperatures. The protein refolded successfully in only one of the 46 simulations, and in that case the refolding pathway mirrored the unfolding pathway at high temperature. In the 45 simulations in which the protein did not fully fold, nonnative salt bridges trapped the protein, which explains why the protein folds relatively slowly from the intermediate state.

Structural and Functional Consequences of Cardiac Troponin C L57Q and I61Q Ca2+-Desensitizing Variants

Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca(2+) binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca(2+) binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca(2+) affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca(2+), but did decrease the interaction in the presence of Ca(2+). In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca(2+). This difference in the absence of Ca(2+) suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca(2+) binding induced by I61Q may result from destabilization of the Ca(2+) binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca(2+) binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca(2+) binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.

Multimolecule test-tube simulations of protein unfolding and aggregation

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.

Promiscuous contacts and heightened dynamics increase thermostability in an engineered variant of the engrailed homeodomain

A thermostabilized variant (UVF) of the engrailed homeodomain (EnHD) was previously engineered by Mayo and co-workers. The melting temperature of the non-natural, designed protein is 50°C higher than the natural wild-type protein (>99 vs. 52°C), and the two proteins share 22% sequence identity. We have performed extensive (1 μs) all-atom, explicit solvent molecular dynamics simulations of the wild-type and engineered proteins to investigate their structural and dynamic properties at room temperature and at 100°C. Our simulations are in good agreement with nuclear magnetic resonance data available for the two proteins [nuclear Overhauser effect crosspeaks (NOEs), J-coupling constants and order parameters for EnHD; and NOEs for UVF], showing that we reproduce the backbone dynamics and side chain packing in the native state of both proteins. UVF was more dynamic at room temperature than EnHD, with respect to both its backbone and side chain motion. When the temperature was raised, the thermostable protein maintained this mobility while retaining its native conformation. EnHD, on the other hand, was unable to maintain its more rigid native structure at higher temperature and began to unfold. Heightened protein dynamics leading to promiscuous and dynamically interchangeable amino acid contacts makes UVF more tolerant to increasing temperature, providing a molecular explanation for heightened thermostability of this protein.