kDa Domain Research Articles

Crystal structure of the N‐terminal domains of the surface cell antigen 4 of Rickettsia

The obligate intracellular, gram-negative bacterium Rickettsia is the causative agent of spotted fevers and typhus in humans. Surface cell antigen (sca) proteins surround these bacteria. We recently reported the co-localization of one of these proteins, sca4, with vinculin in cells at sites of focal adhesions and demonstrated that two vinculin binding sites directed the sca4/vinculin interaction. Here we report the 2.2 Å crystal structure of the conserved N-terminal 38 kDa domain of sca4 from Rickettsia rickettsii. The structure reveals two subdomains. The first is an all-helical domain that is folded in a fashion similar to the dimeric assembly chaperone for rubisco, namely RbcX. The following and highly conserved β-strand domain lacks significant structural similarity with other known structures and to the best of our knowledge represents a new protein fold.

A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Polµ and Polλ

Human DNA polymerases mu (Polµ) and lambda (Polλ) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3′ single-stranded overhangs and trans-polymerization using one 3′ end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr141, Ala142, Cys143, Gln144 and Arg145 in Polµ, and by Trp239, Val240, Cys241, Ala242 and Gln243 in Polλ. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.

The C Terminus of the Catalytic Domain of Type A Botulinum Neurotoxin May Facilitate Product Release from the Active Site

Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a Ki of 4.5 μm, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target.

Magnesium Impacts Myosin V Motor Activity by Altering Key Conformational Changes in the Mechanochemical Cycle

We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1-9 mM) and found that the highest activity occurs at low Mg (0.1-0.3 mM), while there is a 50-60% reduction in activity at high Mg (3-9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence, and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin.ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin.ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase ATPase domain.

Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47(C1) and Mtb-GyrB47(C2), have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P21 and diffracted to resolutions of 2.9 and 3.3 Å, respectively.

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

The removal of initiating primers from the 5'-ends of each Okazaki fragment, required for the generation of contiguous daughter strands, can be catalyzed by the combined action of DNA polymerase δ and Fen1. When the flaps generated by displacement of DNA synthesis activity of polymerase δ become long enough to bind replication protein A or form hairpin structures, the helicase/endonuclease enzyme, Dna2, becomes critical because of its ability to remove replication protein A-coated or secondary structure flaps. In this study, we show that the N-terminal 45-kDa domain of Dna2 binds hairpin structures, allowing the enzyme to target secondary structure flap DNA. We found that this activity was essential for the efficient removal of hairpin flaps by the endonuclease activity of Dna2 with the aid of its helicase activity. Thus, the efficient removal of hairpin structure flaps requires the coordinated action of all three functional domains of Dna2. We also found that deletion of the N-terminal 45-kDa domain of Dna2 led to a partial loss of the intra-S-phase checkpoint function and an increased rate of homologous recombination in yeast. We discuss the potential roles of the N-terminal domain of Dna2 in the maintenance of genomic stability.

Conformationally Trapping the Actin-binding Cleft of Myosin with a Bifunctional Spin Label

We have trapped the catalytic domain of Dictyostelium (Dicty) myosin II in a weak actin-binding conformation by chemically crosslinking two engineered cysteines across the actin-binding cleft, using a bifunctional spin label (BSL). By connecting the lower and upper 50 kDa domains of myosin, the crosslink restricts the conformation of the actin-binding cleft. Crosslinking has no effect on the basal ATPase activity of isolated myosin, but it impairs rigor actin binding and actin-activation of myosin ATPase. EPR spectra of BSL provide insight into actomyosin structural dynamics. BSL is highly immobilized within the actin-binding cleft and is thus exquisitely sensitive to the global orientation and rotational motions of the myosin head. Conventional EPR shows that myosin heads bound to oriented actin filaments are highly disordered with respect to the actin filament axis, in contrast to the nearly crystalline order of myosin heads in rigor. This disorder is similar to that of weakly bound heads induced by ATP, but saturation transfer EPR shows that the disorder of crosslinked myosin is at least 100 times slower. Thus this cleft-crosslinked myosin is remarkably similar, in both actin affinity and rotational dynamics, to SH1-SH2 crosslinked BSL-myosin S1. We conclude that, whether myosin is trapped at the actin-myosin interface or in the force-generating region between the active site and lever arm, the structural state of myosin is intermediate between the weak-binding state preceding phosphate release and the strong-binding state that succeeds it. We propose that it represents the threshold of force generation.

Structural Changes in Myosin Binding Protein C in Different Biochemical Conditions

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filaments of vertebrate striated muscle. Its function in skeletal muscle is not yet understood, but it is known to modulate contractility in the heart. Defects in MyBP-C lead to both skeletal and cardiac muscle disease. MyBP-C is an elongated molecule consisting primarily of a linear array of 10 (skeletal) or 11 (cardiac) immunoglobulin- and fibronectin-like 10 kDa domains. In addition to binding to the thick filament via its C-terminus, MyBP-C can also interact with myosin subfragment-2 and with actin via its N-terminus. MyBP-C is observed in EM images of sarcomeres as a set of up to nine transverse stripes, 43 nm apart, in each half of the A-band. Electron tomography of skeletal muscle in the relaxed state suggests that these stripes are due to extension of MyBP-C from the surface of the thick filament to the thin filaments, to which it binds. To investigate whether MyBP-C might change structurally in different physiological states, we have carried out experiments on isolated myofibrils and A-segments (A-bands free of thin filaments) to determine the appearance of the stripes under different biochemical conditions. Preliminary results suggest that in myofibrils (where thin filaments overlap thick filaments), stripes are present in both relaxed and rigor states, although they are less prominent in rigor. In A-segments (lacking thin filaments), stripes are common in the relaxed and activated (ATP/high Ca2+) states, but weak or absent in rigor. The results are consistent with a model in which the N-terminus of MyBP-C binds to actin filaments when they are available, and to myosin subfragment-2 when they are not.

Multiple Substrate and Domain Binding in Non-Ribosomal Peptide Synthetases

Non-ribosomal peptide synthetases (NRPSs) are enzymatic systems that synthesize important natural products in bacteria and fungi, often with pharmaceutical applications (antibiotics, antitumor agents or immunosuppressants). NRPSs use a series of domains, organized in contiguous modules, to covalently load substrates and condense those loaded on adjacent modules in an assembly line fashion. The substrates may be further modified by tailoring domains. Such multiple catalytic steps require a series of transient, sequential domain/domain and domain/substrate interactions, which are currently poorly understood. We have used NMR to probe and characterize binding sites for several NRPS domains. We show that chemical substrates do interact with NRPS domains and may be involved in modulating domain interactions. We also present the solution structures of hitherto uncharacterized domains and discuss the role of protein dynamics. We introduce novel NMR approaches necessary for such challenging systems (53 kDa domains, dynamic proteins). Understanding NRPS domain communication is a prerequisite to understand the biosynthesis of many natural products and offers prospect for reprogramming NRPS assembly lines.View Large Image | View Hi-Res Image | Download PowerPoint Slide

System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.

Folding of large multidomain proteins by partial encapsulation in the chaperonin TRiC/CCT

The eukaryotic chaperonin, TRiC/CCT (TRiC, TCP-1 ring complex; CCT, chaperonin containing TCP-1), uses a built-in lid to mediate protein folding in an enclosed central cavity. Recent structural data suggest an effective size limit for the TRiC folding chamber of ∼70 kDa, but numerous chaperonin substrates are substantially larger. Using artificial fusion constructs with actin, an obligate chaperonin substrate, we show that TRiC can mediate folding of large proteins by segmental or domain-wise encapsulation. Single or multiple protein domains up to ∼70 kDa are stably enclosed by stabilizing the ATP-hydrolysis transition state of TRiC. Additional domains, connected by flexible linkers that pass through the central opening of the folding chamber, are excluded and remain accessible to externally added protease. Experiments with the physiological TRiC substrate hSnu114, a 109-kDa multidomain protein, suggest that TRiC has the ability to recognize domain boundaries in partially folded intermediates. In the case of hSnu114, this allows the selective encapsulation of the C-terminal ∼45-kDa domain and segments thereof, presumably reflecting a stepwise folding mechanism. The capacity of the eukaryotic chaperonin to overcome the size limitation of the folding chamber may have facilitated the explosive expansion of the multidomain proteome in eukaryotes.

Cloning, expression, crystallization and preliminary X-ray studies of the ferredoxin–NAD(P)+reductase from the thermophilic cyanobacteriumThermosynechococcus elongatusBP-1

Ferredoxin-NADP(+) reductase (FNR) is a flavoenzyme that catalyses the reduction of NADP(+) in the final step of the photosynthetic electron-transport chain. FNR from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TeFNR) contains an additional 9 kDa domain at its N-terminus relative to chloroplastic FNRs and is more thermostable than those from mesophilic cyanobacteria. With the aim of understanding the structural basis of the thermostability of TeFNR and assigning a structural role to the small additional domain, the gene encoding TeFNR with and without an additional domain was engineered for heterologous expression and the recombinant proteins were purified and crystallized. Crystals of TeFNR without the additional domain belonged to space group P2(1), with unit-cell parameters a = 55.05, b = 71.66, c = 89.73 Å, α = 90, β = 98.21, γ = 90°.

DNA stabilization at the Bacillus subtilis PolX core —a binding model to coordinate polymerase, AP-endonuclease and 3′-5′ exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.

Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

Chaperonin Cofactors, Cpn10 and Cpn20, of Green Algae and Plants Function as Hetero-oligomeric Ring Complexes

The chloroplast chaperonin system of plants and green algae is a curiosity as both the chaperonin cage and its lid are encoded by multiple genes, in contrast to the single genes encoding the two components of the bacterial and mitochondrial systems. In the green alga Chlamydomonas reinhardtii (Cr), three genes encode chaperonin cofactors, with cpn10 encoding a single ∼10-kDa domain and cpn20 and cpn23 encoding tandem cpn10 domains. Here, we characterized the functional interaction of these proteins with the Escherichia coli chaperonin, GroEL, which normally cooperates with GroES, a heptamer of ∼10-kDa subunits. The C. reinhardtii cofactor proteins alone were all unable to assist GroEL-mediated refolding of bacterial ribulose-bisphosphate carboxylase/oxygenase but gained this ability when CrCpn20 and/or CrCpn23 was combined with CrCpn10. Native mass spectrometry indicated the formation of hetero-oligomeric species, consisting of seven ∼10-kDa domains. The cofactor "heptamers" interacted with GroEL and encapsulated substrate protein in a nucleotide-dependent manner. Different hetero-oligomer arrangements, generated by constructing cofactor concatamers, indicated a preferential heptamer configuration for the functional CrCpn10-CrCpn23 complex. Formation of heptamer Cpn10/Cpn20 hetero-oligomers was also observed with the Arabidopsis thaliana (At) cofactors, which functioned with the chloroplast chaperonin, AtCpn60α(7)β(7). It appears that hetero-oligomer formation occurs more generally for chloroplast chaperonin cofactors, perhaps adapting the chaperonin system for the folding of specific client proteins.

The Primary DNA-Binding Subsite of the Rat Pol Beta. Energetics of Interactions of the 8-kDa Domain of the Enzyme with the ssDNA

Interactions of the 8-kDa domain of the rat pol β and the intact enzyme with the ssDNA have been studied, using the quantitative fluorescence titration technique. The 8-kDa domain induces large topological changes in the bound DNA structure and engages much larger fragments of the DNA than when embedded in the intact enzyme. The DNA affinity of the domain is predominantly driven by entropy changes, dominated by the water release from the protein. The thermodynamic characteristics dramatically change when the domain is embedded in the intact polymerase, indicating the presence of significant communication between the 8-kDa domain and the catalytic 31-kDa domain. The diminished water release from the 31-kDa domain strongly contributes to its dramatically lower DNA affinity, as compared to the 8-kDa domain. Unlike the 8-kDa domain, the DNA binding of the intact pol β is driven by entropy changes, originating from the structural changes of the formed complexes.

Modulating Contraction by Binding of MyBP-C to Actin

Myosin-binding protein C (MyBP-C) is a ∼130 kDa protein of the thick filaments of vertebrate skeletal and cardiac muscle. It consists of a linear array of ten or eleven globular, 10-kDa domains from the immunoglobulin (Ig) and fibronectin type III families, and an additional, MyBP-C-specific motif. The cardiac isoform, cMyBP-C, plays a key role in modulating cardiac function, and mutations in MyBP-C cause heart disease. Despite its discovery 40 years ago, the mechanism of MyBP-C function remains poorly understood. In vitro studies suggest that it could modulate contraction by binding to thin filaments, but there has been no evidence for this in situ. We used electron tomography of exceptionally well-preserved skeletal muscle to study the 3D organization of MyBP-C in the intact sarcomere. The tomogram shows that MyBP-C projects perpendicular to the thick filament surface and reaches neighboring thin filaments. This thick-thin filament bridge suggests a possible physical basis for modulating filament sliding and thus contraction. In vitro, binding to actin has been shown to occur via MyBP-C's N-terminal end. To understand the structural basis of this binding, we used negative stain electron microscopy and 3D reconstruction to study F-actin decorated with bacterially expressed N-terminal cMyBP-C fragments. Clear decoration was obtained under a variety of salt conditions. 3D reconstructions showed MyBP-C density starting over subdomain 1 of actin and extending tangentially towards actin's pointed end. Molecular fitting with an atomic structure of a MyBP-C Ig domain suggested that most of the N-terminal domains may be well ordered on actin. The location of binding was such that it appeared to overlap the relaxed (low Ca2+) position of tropomyosin but not the activated position. This suggests that MyBP-C might help determine the state of thin filament activity by modulating tropomyosin position on actin.

【Original Contribution】 Characterization of Mouse Monoclonal Antibodies to Human Protein 4.1R FERM Domain: Epitope Mapping and Application to FERM Domain Binding to Red Blood Cell Inside-out Vesicles

In human erythrocytes, the 80-kDa isoform of protein 4.1R, 4.1R80, maintains mechanical membrane stability and deformability as a result of multiple protein-protein interactions. 4.1R80 binds to transmembrane proteins glycophorin C (GPC) and band 3 via its 30-kDa N-terminal FERM (Four one-Ezrin-Radixin-Moesin) domain (referred to as “R30” in the present study) and to spectrin and actin via a 10-kDa domain. Although the sites in R30 responsible for interaction with transmembrane proteins have been extensively studied, the identity of these sites has been challenged recently. Antibodies, in particular monoclonal antibodies (mAbs), are powerful tools not only for immunochemical studies but also for functional analyses such as the monitoring of the dynamic interactions of R30 with its binding partners. In the present study, we have generated mouse mAbs against recombinant R30 protein, and characterized their respective recognition epitopes in R30 using various recombinant proteins. Four representative clones, #5, #7, #9, #13 recognized the Y131DPELHGVDYVSDFKLAPN (pep8.1), Q150 TKELEEKVMELHKSYR (pep8.2), M1HCKVSLLDDTVYECVVE (pep4) and Q247EQYESTIGFKLPSYRA (pep13) epitopes, respectively. These sequences are located in the N-,α- and C-lobe structure of R30, respectively. IAsys-based in vitro binding analyses enabled us to demonstrate that the binding of R30 to pH 11-treated inside-out-vesicles (IOVs) was reduced by two combinations of mAbs (#5 and #9 or #7 and #9) but not by any of the mAbs alone and to confirm that the GPC binding site of R30 was located at or near pep8.1 and pep8.2 in the α- lobe of R30. Our study validates that this novel panel of mAbs constitutes a powerful tool for various types of analyses of 4.1R.

Allosteric Control of Cofactor Binding to Nuclear Hormone Receptors

The nuclear receptor superfamily (NRs) comprises ligand-dependent transcription regulators, many of which function as homodimers or heterodimers with RXR. We show that addition of an SRC-1 coactivator-derived peptide to monomeric liganded retinoic acid receptor (RAR) promotes efficient homodimer formation. X-ray crystallography revealed that homodimers are asymmetrical, reminiscent of functional RXR-RAR heterodimers with distinct conformations of ligand in the two subunits. A global switch to the dimeric state with a 2RAR:1SRC-1 stoichiometry was also seen using a larger 20kDa domain of SRC-1. Our study reveals an allosteric mechanism whereby coactivator binding to one RAR partner affects ligand conformation and H12 position of the second partner, hrough the dimer interface. A similar control on the recruitment of SRC-1 is also observed with ER in the crystal structures of ERa bound to agonist ligands and coactivator peptide.

Magnesium Regulates Myosin V Motor Activity by Altering Key Conformational Changes in the Nucleotide Binding Pocket

We investigated how magnesium impacts key conformational changes in the nucleotide binding pocket of myosin V and how these alterations impact the mechanochemical cycle. The conformation of the nucleotide binding pocket was examined using our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. Our previous work demonstrated the rate limiting conformation change in the actomyosin V ATPase cycle is opening of the nucleotide binding pocket which precedes ADP release from the open state. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free magnesium concentrations (0-10 mM) and find that the highest activity occurs at 0.5 mM magnesium, while there is a 50-60% reduction in activity above 4 mM magnesium. We also demonstrate that the motor activity assayed by in vitro motility is similarly dependent on magnesium concentrations. Transient kinetic studies of mantADP binding/release with actomyosin V FlAsH demonstrate the equilibrium between the open and closed nucleotide binding pocket conformations is dependent on magnesium with the closed state stabilized by magnesium. We find that the kinetics of the nucleotide binding pocket opening step correlates well with the ATPase and motility results over a wide range of magnesium concentrations. In the absence of magnesium (presence of 4 mM EDTA) the nucleotide binding pocket populates a single conformation that is dramatically open at higher temperatures. In addition, magnesium significantly slows the rate of ADP release from the open state. Our results shed light on the structural mechanism of ADP release in myosin V and allow us to speculate about the conserved conformational pathways involved in strain sensitive ADP release in myosins.