Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor’s function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.

The [Mg(2+)] dependence of ADP binding to myosin V and actomyosin V was measured from the fluorescence of mantADP. Time courses of MgmantADP dissociation from myosin V and actomyosin V are biphasic with fast observed rate constants that depend on the [Mg(2+)] and slow observed rate constants that are [Mg(2+)]-independent. Two myosin V-MgADP states that are in reversible equilibrium, one that exchanges nucleotide and cation slowly (strong binding) and one that exchanges nucleotide and cation rapidly (weak binding), account for the data. The two myosin V-MgADP states are of comparable energies, as indicated by the relatively equimolar partitioning at saturating magnesium. Actin binding lowers the affinity for bound Mg(2+) 2-fold but shifts the isomerization equilibrium approximately 6-fold to the weak ADP binding state, lowering the affinity and accelerating the overall rate of MgADP release. Actin does not weaken the affinity or accelerate the release of cation-free ADP, indicating that actin and ADP binding linkage is magnesium-dependent. Myosin V and myosin V-ADP binding to actin was assayed from the quenching of pyrene actin fluorescence. Time courses of myosin V-ADP binding and release are biphasic, consistent with the existence of two (weak and strong) quenched pyrene actomyosin V-ADP conformations. We favor a sequential mechanism for actomyosin V dissociation with a transition from strong to weak actin-binding conformations preceding dissociation. The data provide evidence for multiple myosin-ADP and actomyosin-ADP states and establish a kinetic and thermodynamic framework for defining the magnesium-dependent coupling between the actin and nucleotide binding sites of myosin.

Continuum methods were used to calculate the electrostatic contributions of charged and polar side chains to the overall stability of a small 41-residue helical protein, the peripheral subunit-binding domain. The results of these calculations suggest several residues that are destabilizing, relative to hydrophobic isosteres. One position was chosen to test the results of these calculations. Arg8 is located on the surface of the protein in a region of positive electrostatic potential. The calculations suggest that Arg8 makes a significant, unfavorable electrostatic contribution to the overall stability. The experiments described in this paper represent the first direct experimental test of the theoretical methods, taking advantage of solid-phase peptide synthesis to incorporate approximately isosteric amino acid substitutions. Arg8 was replaced with norleucine (Nle), an amino acid that is hydrophobic and approximately isosteric, or with alpha-amino adipic acid (Aad), which is also approximately isosteric but oppositely charged. In this manner, it is possible to isolate electrostatic interactions from the effects of hydrophobic and van der Waals interactions. Both Arg8Nle and Arg8Aad are more thermostable than the wild-type sequence, testifying to the validity of the calculations. These replacements led to stability increases at 52.6 degrees C, the T(m) of the wild-type, of 0.86 and 1.08 kcal mol(-)(1), respectively. The stability of Arg8Nle is particularly interesting as a rare case in which replacement of a surface charge with a hydrophobic residue leads to an increase in the stability of the protein.

Intracellular trafficking depends on the docking and fusion of transport vesicles with cellular membranes. Central to docking and fusion is the pairing of SNARE proteins (soluble NSF attachment protein receptors) associated with the vesicle and target membranes (v- and t-SNAREs, respectively). Here, the X-ray structure of an N-terminal conserved domain of the neuronal t-SNARE syntaxin-1A was determined to a resolution of 1.9 A using multiwavelength anomalous diffraction. This X-ray structure, which is in general agreement with an NMR structure of a similar fragment, provides new insight into the interaction surface between the N-terminal domain and the remainder of the protein. In vitro characterization of the intact cytoplasmic domain of syntaxin revealed that it forms dimers, and probably tetramers, at low micromolar concentrations, with concomitant structural changes that can be detected by limited proteolysis. These observations suggest that the promiscuity characteristic of pairing between v-SNAREs and t-SNAREs extends to the formation of homo-oligomeric t-SNARE complexes as well. They also suggest a potential role for the neuronal Sec1 protein (nSec1) in preventing the formation of syntaxin multimers.

We have measured the energetics of ATP and ADP binding to single-headed actomyosin V and VI from the temperature dependence of the rate and equilibrium binding constants. Nucleotide binding to actomyosin V and VI can be modeled as two-step binding mechanisms involving the formation of collision complexes followed by isomerization to states with high nucleotide affinity. Formation of the actomyosin VI-ATP collision complex is much weaker and slower than for actomyosin V. A three-step binding mechanism where actomyosin VI isomerizes between two conformations, one competent to bind ATP and one not, followed by rapid ATP binding best accounts for the data. ADP binds to actomyosin V more tightly than actomyosin VI. At 25°C, the strong ADP-binding equilibria are comparable for actomyosin V and VI, and the different overall ADP affinities arise from differences in the ADP collision complex affinity. The actomyosin-ADP isomerization leading to strong ADP binding is entropy driven at >15°C and occurs with a large, positive change in heat capacity (∆C P°) for both actomyosin V and VI. Sucrose slows ADP binding and dissociation from actomyosin V and VI but not the overall equilibrium constants for strong ADP binding, indicating that solvent viscosity dampens ADP-dependent kinetic transitions, presumably a tail swing that occurs with ADP binding and release. We favor a mechanism where strong ADP binding increases the dynamics and flexibility of the actomyosin complex. The heat capacity (∆C P°) and entropy (∆S°) changes are greater for actomyosin VI than actomyosin V, suggesting different extents of ADP-induced structural rearrangement.Myosins are molecular motors that utilize the energy from adenosine 5′-triphosphate (ATP) 1 binding, hydrolysis, and product release to perform mechanical work along actin filament tracks for diverse physiological processes including muscle contraction, cell division, vesicle and RNA transport, and chemotaxis (1, 2). Force generation and motility (work output) are coupled to chemical catalysis. ATP binding and hydrolysis cycle myosin vectorially through a series of energetic (conformational) states that bind actin filaments strongly (attached) or weakly (detached) depending on the chemical state of the bound nucleotide. In the absence of nucleotide or with bound adenosine 5′-diphosphate (ADP), myosin binds actin filaments strongly (strong binding states). With ATP and the hydrolysis products, ADP and phosphate (P i ), bound myosin binds actin weakly (weak binding states). The fraction of the catalytic cycle time spent strongly attached to actin is referred to as the duty ratio.Although all characterized myosins share a common ATPase cycle pathway, detailed kinetic analyses indicate that modulation of the rate and equilibrium constants that define the ATPase cycle confer specific properties to the motor for specific physiological tasks (3). Some myosins are tuned for anchoring cellular structures and maintaining tension, while others are better suited for transporting cargo or generat...

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A long-standing question has focused on how GR and other receptors precisely control gene expression. One difficulty in addressing this is that GR function is influenced by multiple factors including ligand and coactivator levels, chromatin state, and allosteric coupling. Moreover, the receptor recognizes an array of DNA sequences that generate a range of transcriptional activities. Such complexity suggests that any single parameter-DNA binding affinity, for example-is unlikely to be a dominant contributor to function. Indeed, a number of studies have suggested that for GR and other receptors, binding affinity toward different DNA sequences is poorly correlated with transcriptional activity. As a step toward determining the factors most predictive of GR function, we rigorously examined the relationship between in vitro GR-DNA binding energetics and in vivo transcriptional activity. We first demonstrate that previous approaches for assessing affinity-function relationships are problematic due to issues of data transformation and linearization. Thus, the conclusion that binding energetics and transcriptional activity are poorly correlated is premature. Using more appropriate analyses, we find that energetics and activity are in fact highly correlated. Furthermore, this correlation can be quantitatively accounted for using simple binding models. Finally, we show that the strong relationship between energetics and transcriptional activity is recapitulated in multiple promoter contexts, cell lines, and chromatin environments. Thus, despite the complexity of GR function, DNA binding energetics are the primary determinant of sequence-specific transcriptional activity.

The glucocorticoid receptor (GR) is a member of the steroid receptor family of ligand-activated transcription factors. A number of studies have shown that steroid receptors regulate distinct but overlapping sets of genes; however, the molecular basis for such specificity remains unclear. Previous work from our laboratory has demonstrated that under identical solution conditions, three other steroid receptors – the progesterone receptor A-isoform (PR-A), the progesterone receptor B-isoform (PR-B), and estrogen receptor-α (ER-α) – differentially partition their self-association and promoter binding energetics. For example, PR-A and PR-B generate similar dimerization free energies but differ significantly in their extents of inter-site cooperativity. Conversely, ER-α maintains inter-site cooperativity most comparable to PR-A, yet dimerizes with an affinity orders of magnitude greater than either of the PR isoforms. We have speculated that these differences serve to generate receptor-specific promoter occupancies, and thus receptor-specific gene regulation. Noting that GR regulates a unique subset of genes relative to the other receptors, we hypothesized that the receptor should maintain a unique set of interaction energetics. We rigorously determined the self-association and promoter binding energetics of full-length, human GR under conditions identical to those used in our earlier studies. We find that unlike all other receptors, GR shows no evidence of reversible self-association. Moreover, GR assembles with strong inter-site cooperativity comparable to that seen only for PR-B. Finally, simulations show that such partitioning of interaction energetics allows for receptor-specific promoter occupancies, even under conditions where multiple receptors are competing for binding at identical sites.

Parasites from the genus Plasmodium are the causative agents of malaria. The mobility, infectivity, and ultimately pathogenesis of Plasmodium falciparum rely on a macromolecular complex, called the glideosome. At the core of the glideosome is an essential and divergent Myosin A motor (PfMyoA), a first order drug target against malaria. Here, we present the full-length structure of PfMyoA in two states of its motor cycle. We report novel interactions that are essential for motor priming and the mode of recognition of its two light chains (PfELC and MTIP) by two degenerate IQ motifs. Kinetic and motility assays using PfMyoA variants, along with molecular dynamics, demonstrate how specific priming and atypical sequence adaptations tune the motor’s mechano-chemical properties. Supported by evidence for an essential role of the PfELC in malaria pathogenesis, these structures provide a blueprint for the design of future anti-malarials targeting both the glideosome motor and its regulatory elements.