kDa Domain Research Articles

Identification of Novel Protein Kinase A Phosphorylation Sites in the M-domain of Human and Murine Cardiac Myosin Binding Protein-C Using Mass Spectrometry Analysis

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.

Analysis of Thermal Stability of Protein 4.1R FERM Domain

[Motivation and Aim]The crystal structure of N-terminal 30kDa domain of protein 4.1R (R30, “FERM” domain), that is a membrane skeletal protein, is three-lobe-clover. The transmembrane proteins, Glycophorin C (GPC) and band 3, and p55 bind to each different lobe of R30. Calmodulin (CaM) also binds to R30 in Ca2+-independent manner. Binding with these proteins may stabilize R30. In the present study, we analyzed temperature dependent changes of R30 structure and its binding affinity to apo-CaM.[Materials and Methods]1) The recombinant proteins (cytoplasmic domains of GPC and band 3, p55, and R30) were purified as GST fusion protein from bacteria lysate. CaM was purified from bovine brain.2) FT-IR (attenuated total reflection (ATR) analysis), with Tensor27 and BIO-ATRII accessory (Bruker Optics K.K.) was used for detecting of secondary structure of R30.3) Dynamic light scattering (DLS) analysis was carried out with Zetasizer Nano ZS® (Sysmex Corp.).4) The binding kinetics of R30 to proteins was analyzed using IAsys (Affinity Sensors). R30 dissolved in 10mM HEPES, pH7.4 containing 0.1M NaCl and 1mM EDTA was incubated at 4°C∼60°C for 30 min and the binding activity was measured.[Results]1) ATR analysis of R30 showed dramatic increase in intensity of β-sheet (1628cm−1 and 1672cm−1) with increase in temperature from 40°C to 45°C. The corresponding change was small in the presence of apo-CaM.2) In DLS measurement, R30 became to be aggregated around 45°C.3) R30 denatured at 50°C lost binding ability to apo-CaM, cytoplasmic domains of GPC and band 3. The binding ability of R30 to p55 did at 40°C.[Discussion]Aggregation of R30 at 45°C may be caused through its β-sheet. FT-IR results suggested increasing of intramolecular β-sheet. Actually, p55 binding site is located at the β-sheet structure rich domain.

Kinetics and Thermodynamics of Nucleotide Binding Pocket Opening/closing in Myosin V Monitored with FRET

Kinetic and structural studies of both muscle and non-muscle myosins have revealed that the enzymatic cycle of these motors frequently contains more than one actomyosin ADP state. Interestingly, the rate of ADP release in myosin motors is thought to be the main determinant of sliding velocity in muscle, suggesting strain dependent ADP release may be a critical mechanism of mechanochemical coupling. Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH (MV FlAsH) can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides. We also determined that this donor-acceptor pair likely monitors opening/closing of the nucleotide binding pocket. Currently, we utilized the FRET signal to examine the kinetics of nucleotide binding pocket opening during the process of mantADP release from acto-MV FlAsH. We obtained evidence that the nucleotide binding pocket goes from a closed to an open conformation prior to the release of ADP. We also explored the temperature dependence of the closed to open transition and nucleotide release steps. We find that at lower temperatures the closed conformation is favored while at higher temperature the open conformation is favored. The more rapid ADP release step which follows nucleotide binding pocket opening is also temperature dependent. Therefore, since both steps are temperature-dependent they likely require significant conformational changes. We also compared our FRET results to the rate of ATP-induced dissociation from actin in the presence of ADP monitored by light scatter. Understanding how strain alters either of these two steps may be critical for elucidating the structural mechanism of strain-dependent ADP release in myosins.

Coupling the Actin Binding Cleft and Nucleotide Binding Pocket in Myosin V

Previously we have demonstrated in fluorescence resonance energy transfer (FRET) studies that mant labeled nucleotides and IAEDANS actin can act as good donor probes for a FlAsH labeled acceptor site in the upper 50 kDa domain of myosin V. We examined the temperature dependence of the FRET signal between mantADP and MV WT FlAsH in the presence and absence of actin. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-35°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. However, the mutant E442A, which is incapable of hydrolyzing ATP, remains in a high FRET state (closed pocket) with mantATP bound in the presence or absence of actin. Our results suggest a more flexible conformation of myosin in the presence of ADP compared to ATP which allows myosin to populate two actomyosin.ADP state conformations. These results are supported by the lifetime FRET analysis, and by computational FIRST/FRODA analysis of the intrinsic flexibility found in different x-ray crystal structures. We also plan to explore the temperature dependent conformational dynamics of the actin binding cleft using the IAEDANS actin (donor) and MV FlAsH (acceptor) pair in the presence of ATP, ADP, and absence of nucleotide using steady state and lifetime based FRET measurements. Our results will provide critical insights into the mechanocoupling that may occur between the nucleotide-binding pocket and actin binding cleft in myosin motors.

Fragile histidine triad protein: structure, function, and its association with tumorogenesis

The human fragile histidine triad (FHIT) gene is a putative tumor suppressor gene, which is located at chromosome region 3p14.2. It was suggested that the loss of heterozygosity (LOH), homozygous deletions, and abnormal expression of the FHIT gene were involved in several types of human malignancies. To determine the role of FHIT in various cancers, we have performed structural and functional analysis of FHIT in detail. The protein FHIT catalyzes the Mg(2+) dependent hydrolysis of P1-5 cent-O-adenosine-P3-5 cent-O-adenosine triphosphate, Ap3A, to AMP, and ADP. The reaction is thought to follow a two-step mechanism. Histidine triad proteins, named for a motif related to the sequence H-cent-H-cent-H-cent-cent- (cent, a hydrophobic amino acid), belong to superfamily of nucleotide hydrolases and transferases. This enzyme acts on the R-phosphate of ribonucleotides, and contain a approximately 30-kDa domain that is typically a homodimer of approximately 15 kDa polypeptides with catalytic site. Here we have gathered information is known about biological activities of FHIT, the structural and biochemical bases for their functions. Our approach may provide a comparative framework for further investigation of FHIT.

Natively unfolded nucleic acid binding P8 domain of SeMV polyprotein 2a affects the novel ATPase activity of the preceding P10 domain

Open reading frame (ORF) 2a of Sesbania mosaic virus (SeMV) codes for polyprotein 2a (Membrane anchor-protease-VPg-P10–P8). The C-terminal domain of SeMV polyprotein 2a was cloned, expressed and purified in order to functionally characterize it. The protein of size 8kDa (P8) domain, like viral protein genome linked (VPg), was found to be natively unfolded and could bind to nucleic acids. Interestingly, P10–P8 but not P8 showed a novel Mg2+ dependent ATPase activity that was inhibited in the presence of poly A. In the absence of P8, the ATPase activity of the protein of size 10kDa (P10) domain was reduced suggesting that the natively unfolded P8 domain influenced the P10 ATPase.

Anchorage of Vinculin to Lipid Membranes Influences Cell Mechanical Properties

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944–978) and the unstructured C-terminal arm (residues 1052–1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(−/−) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinΔC, residues 1–1051). MEF-vinΔC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinΔC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinΔC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.

Cell Death and Learning Impairment in Mice Caused by in Vitro Modified Pro-NGF Can Be Related to Its Increased Oxidative Modifications in Alzheimer Disease

Pro-nerve growth factor (pro-NGF) is expressed at increased levels in Alzheimer's disease (AD)-affected brains and is able to induce cell death in cultures; however, the reasons for these phenomena remain elusive. Here we show that pro-NGF in human AD-affected hippocampus and entorhinal cortex is modified by advanced glycation and lipoxidation end-products in a stage-dependent manner. These modifications block pro-NGF processing to mature NGF, thus making the proneurotrophin especially effective in inducing apoptosis of PC12 cells in culture through the p75 neurotrophin receptor. The processing of advanced glycation and lipoxidation end-products in vitro modified recombinant human pro-NGF is severely impaired, as evidenced by Western blot and by examining its physiological functionality in cell cultures. We also report that modified recombinant human pro-NGF, as well as pro-NGF isolated from human brain affected by AD, cause impairment of learning tasks when administered intracerebroventricularly in mice, which correlates with AD-associated learning impairment. Taken together, the data we present here offer a novel pathway of ethiopathogenesis in AD caused by advanced glycation and lipoxidation end-products modification of pro-NGF.

Conformational Stability and DNA Binding Specificity of the Cardiac T-Box Transcription Factor Tbx20

The transcription factor Tbx20 acts within a hierarchy of T-box factors in lineage specification and morphogenesis in the mammalian heart and is mutated in congenital heart disease. T-box family members share a approximately 20-kDa DNA-binding domain termed the T-box. The question of how highly homologous T-box proteins achieve differential transcriptional control in heart development, while apparently binding to the same DNA sequence, remains unresolved. Here we show that the optimal DNA recognition sequence for the T-box of Tbx20 corresponds to a T-half-site. Furthermore, we demonstrate using purified recombinant domains that distinct T-boxes show significant differences in the affinity and kinetics of binding and in conformational stability, with the T-box of Tbx20 displaying molten globule character. Our data highlight unique features of Tbx20 and suggest mechanistic ways in which cardiac T-box factors might interact synergistically and/or competitively within the cardiac regulatory network.

Alternative Exon 9-Encoded Relay Domains Affect More than One Communication Pathway in the Drosophila Myosin Head

We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K1k+2) and ADP affinity (KAD), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.

Solution structure and glycophorin C binding studies of the protein 4.1R FERM α‐lobe domain

Red blood cell protein 4.1 (4.1R, an 80 kDa isoform),1 one of the major erythrocyte cytoskeletal proteins, forms two major ternary complexes in red blood cells, 4.1Ractin-spectrin and 4.1R-p55-glycophorin C (GPC), and plays important roles in maintaining cell morphology and membrane mechanical properties.1–7 This protein consists of four functional domains: the FERM (4.1 and ezrin/radixin/moesin)8 domain (also termed the N-terminal 30 kDa membrane-binding domain), the 16 kDa regulatory domain, the spectrin-actin-binding (SAB) domain, and the C-terminal 22/24 kDa domain. The 4.1R FERM domain is a multifunctional domain that forms a cloverleaf-like fold containing three structural and functional domains (N-lobe, a-lobe, and C-lobe domains).9 In the 4.1R-p55-GPC ternary complex, the 4.1R FERM a-lobe domain interacts with the N-terminal cytoplasmic domain of GPC, the 4.1R FERM C-lobe10 and/or N-lobe domain11 interacts with the p55 HOOK domain, and the p55 PDZ domain interacts with the C-terminal cytoplasmic domain of GPC.10,12–14 We previously examined the specific binary interaction between the p55 PDZ domain and the C-terminal cytoplasmic domain of GPC by NMR, as the first step to obtain structural insights into the 4.1R-p55-GPC ternary complex.15,16 Our NMR studies clearly revealed how the p55 PDZ domain interacts with two key C-terminal residues, Tyr126 and Ile128, of GPC at the atomic level.16 We have now focused on the binary interaction between the 4.1R FERM a-lobe domain and the N-terminal cytoplasmic domain of GPC, as the second step toward our goal. In the binary interaction between 4.1R and GPC, the 4.1R-binding site on GPC reportedly corresponds to the Nterminal 12 amino acid residues of its cytoplasmic domain (residues 82–93), and the RHK (residues Arg86-His87Lys88) motif is probably crucial for binding the 4.1R FERM a-lobe domain.13 In contrast, the GPC-binding site on the 4.1R FERM domain reportedly corresponds to the sequence encoded by exon 8 (residues Tyr94-Arg166) in the 4.1R alobe domain, and the ELEE motif (residues Glu153Glu156) is considered to be a good candidate for the GPC-binding site.9,10 However, there is no experimental evidence that the ELEE motif contributes to the interaction with the N-terminal cytoplasmic domain of GPC. To better understand this interaction mode, we prepared the 4.1R FERM a-lobe domain (residues Asp83-Tyr187) and performed an NMR analysis of this domain with the N-terminal cytoplasmic domain of GPC. Here, we report the first NMR-derived structure of the 4.1R FERM a-lobe domain and propose a new GPC-binding site on the 4.1R FERM a-lobe domain, based on our NMR experiments.

Single molecule measurements of ATP-myosin V and ADP-myosin V

We investigate the conformations of myosin V bound to ATP and to ADP via single molecule FRET measurements. The myosin V is labeled with FlAsH in the upper 50kDa domain, and the bound nucleotides are labeled with Rhodamine 101. We have carried out two types of single molecule FRET measurements on this complex: 1) we record the transit of single molecules diffusing through the focal region of a probe laser (473 nm); 2) we record the time trajectory of each molecule while it is encapsulated within an optically trapped femtoliter aqueous nanodroplet (hydrosome).

Temperature Dependent Energy Transfer Measurements Reveal Flexibility in the Upper 50 kDa Domain of Myosin V

Our previous work has demonstrated that labeling myosin V in the upper 50 kDa domain with the biarsenical dye FlAsH can serve as an acceptor for fluorescence resonance energy transfer studies with mant labeled nucleotides and IAEDANS actin. These FRET studies suggest that myosin V can adopt a conformation in which the nucleotide binding pocket and the actin binding cleft are in a closed conformation. Our studies suggest the upper 50 kDa domain may be highly flexible in certain nucleotide-states which allows tight binding to nucleotide and actin. Molecular geometric simulations demonstrate the upper 50 kDa domain is most flexible in the myosin V.ADP state, consistent with this state having a high affinity for ADP and actin. Currently, we examined the temperature dependence of the FRET signal between mantADP and MV FlAsH. We found that at low temperature (4-15°C) a high FRET state dominates (closed pocket) while at high temperature (30-37°C) a low FRET state dominates (open pocket). This transition is reversible suggesting a temperature-dependent conformational change. We also found that FlAsH labeled G440A MV, a non-hydrolyzable mutant, has a similar temperature-dependent transition in the presence of mantATP. In contrast, the transition does not occur in the presence of mantADP.BeFx or with the non-hydrolyzable E44A MV mutant in the presence of mantATP. Our results suggest coordination of the gamma-phosphate of ATP rigidifies the upper 50 kDa domain which results in a weak actin affinity state (open actin binding cleft and closed nucleotide binding pocket). However, upon phosphate release the upper 50kDa domain becomes more flexible which allows myosin to adopt a conformation in which it has a high affinity for both nucleotide and actin (closed nucleotide binding pocket and actin binding cleft).

Structure-Function Studies of Myosin Motor Domains

Domain insertions and replacement of functional modules in the myosin head fragment with synthetic sequences provide efficient means to manipulate key features of the myosin motor such as actin- and nucleotide-affinity, coupling between the actin- and nucleotide-binding sites, force production and even the direction of movement in a well defined manner. Additional, this approach facilitates the production of functional motor domains derived from a wide range of members of the myosin family. In recent work, we have combined this approach with the use of small molecule effectors of myosin motor activity. We identified pentabromopseudilin (PBP) and related halogenated alkaloids as potent inhibitors of myosin-dependent processes such as isometric tension development, unloaded shortening velocity, and in vitro motility. Coupling between the actin and nucleotide binding sites is reduced in the presence of these inhibitors. PBP-induced changes in rate constants for ATP-binding, ATP-hydrolysis and ADP dissociation extend the time required per actin-activated myosin ATPase cycle. Additionally, the ratio of time spent per ATPase cycle in strong and weak binding states is shifted by PBP and related compounds in favor of non-force generating states. To elucidate the binding mode of these compounds, we crystallized their complexes formed with the Dictyostelium myosin-2 motor domain. In every case, the electron density for the small molecule inhibitor is unambiguous. All compounds bind to a novel allosteric site near actin-binding residues at the tip of the 50-kDa domain. The residues involved in the binding of this new class of inhibitors are only moderately conserved between the members of the different myosin classes. This is consistent with the observed differences in IC50 values. The results of molecular modeling studies show that these isoform-specific variations in the extent of inhibition can be predicted at least in trend for each of the new compounds.

Myosin II Trapped In A Weak Actin-binding State Through A Chemical Crosslink Across The Actin-Binding Cleft

We have trapped Dictyostelium myosin II in a weak actin-binding conformation by chemically crosslinking two engineered Cys across the actin-binding cleft using a bifunctional spin label (BSL). With sites in the lower and upper 50 kDa domains, the crosslink restricts the conformation of the actin-binding cleft. The crosslinking reaction was monitored by electron paramagnetic resonance (EPR) based on the spin label immobilization that occurs upon reaction of both Cys. The EPR spectrum of crosslinked myosin is sensitive to structural changes induced by both nucleotide- and actin-binding. Functional assays demonstrate that crosslinking partially impairs actin binding and actin-activation but has negligible effects on basal ATPase activity. We propose that crosslinked myosin is trapped in a weak actin-binding structure in which phosphate release is inhibited by the presence of actin. This conformation presumably binds actin weakly but cannot transition to the “closed” cleft structure that is populated with strong actin-binding (Klein et al, 2008, PNAS 105:12867-72). The weak actin-binding structure has proven difficult to characterize because of its transient nature; BSL-crosslinked myosin provides a stable model system for analysis of structural dynamics. We are using EPR to analyze the orientation of BSL-crosslinked myosin attached to actin in skinned muscle fibers, and we are using nucleotide probes to investigate the coupling between the actin-binding cleft and the nucleotide-binding pocket. This work is complementary to a study in which BSL was used to crosslink SH1 (C707) and SH2 (C697) in the force-generating domain of myosin, producing a stable complex that bound weakly to actin with slow orientational disorder (Thompson et al., 2008, Biophys. J., in press). This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute.

The mechanism of pentabromopseudilin inhibition of myosin motor activity

We have identified pentabromopseudilin (PBP) as a potent inhibitor of myosin-dependent processes such as isometric tension development and unloaded shortening velocity. PBP-induced reductions in the rate constants for ATP binding, ATP hydrolysis and ADP dissociation extend the time required per myosin ATPase cycle in the absence and presence of actin. Additionally, coupling between the actin and nucleotide binding sites is reduced in the presence of the inhibitor. The selectivity of PBP differs from that observed with other myosin inhibitors. To elucidate the binding mode of PBP, we crystallized the Dictyostelium myosin-2 motor domain in the presence of Mg(2+)-ADP-meta-vanadate and PBP. The electron density for PBP is unambiguous and shows PBP to bind at a previously unknown allosteric site near the tip of the 50-kDa domain, at a distance of 16 A from the nucleotide binding site and 7.5 A away from the blebbistatin binding pocket.

The inhibitory action of kohamaic acid A derivatives on mammalian DNA polymerase beta.

We previously isolated a novel natural product, designated kohamaic acid A (KA-A, compound 1), as an inhibitor of the first cleavage of fertilized sea urchin eggs, and found that this compound could selectively inhibit the activities of mammalian DNA polymerases (pols). In this paper, we investigated the structure and bioactivity of KA-A and its chemically synthesized 11 derivatives (i.e., compounds 2–12), including KA-A - fatty acid conjugates. The pol inhibitory activity of compound 11 [(1S*,4aS*,8aS*)-17-(1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethyl-naphthalen-1-yl)heptadecanoic acid] was the strongest among the synthesized compounds, and the range of IC50 values for mammalian pols was 3.22 to 8.76 μM; therefore, the length of the fatty acid side chain group of KA-A is important for pol inhibition. KA-A derivatives could prevent human cancer cell (promyelocytic leukemia cell line, HL-60) growth with the same tendency as the inhibition of mammalian pols. Since pol β is the smallest molecule, we used it to analyze the biochemical relationship with KA-A derivatives. From computer modeling analysis (i.e., docking simulation analysis), these compounds bound selectively to four amino acid residues (Leu11, Lys35, His51 and Thr79) of the N-terminal 8-kDa domain of pol β, and the binding energy between compound 11 and pol β was largest in the synthesized compounds. The relationship between the three-dimensional molecular structures of KA-A-related compounds and these inhibitory activities is discussed.

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanA.

Streptococcus pneumoniae genomes encode three sialidases, NanA, NanB and NanC, which are key virulence factors that remove sialic acids from various glycoconjugates. The enzymes have potential as drug targets and also as vaccine candidates. The 115 kDa NanA is the largest of the three sialidases and is anchored to the bacterial membrane. Although recombinantly expressed full-length NanA was soluble, it failed to crystallize; therefore, a 56.5 kDa domain that retained full enzyme activity was subcloned. The purified enzyme was crystallized in 0.1 M MES pH 6.5, 30%(w/v) PEG 4000 using the sitting-drop vapour-diffusion method. Data were collected at 100 K to 2.5 A resolution from a crystal grown in the presence of the inhibitor 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.2, b = 95.6, c = 226.6 A. The structure was solved by molecular replacement and refined to final R and R(free) factors of 0.246 and 0.298, respectively.

Structural insights on the pamoic acid and the 8 kDa domain of DNA polymerase beta complex: towards the design of higher-affinity inhibitors.

BackgroundDNA polymerase beta (pol beta), the error-prone DNA polymerase of single-stranded DNA break repair as well as base excision repair pathways, is overexpressed in several tumors and takes part in chemotherapeutic agent resistance, like that of cisplatin, through translesion synthesis. For this reason pol beta has become a therapeutic target. Several inhibitors have been identified, but none of them presents a sufficient affinity and specificity to become a drug. The fragment-based inhibitor design allows an important improvement in affinity of small molecules. The initial and critical step for setting up the fragment-based strategy consists in the identification and structural characterization of the first fragment bound to the target.ResultsWe have performed docking studies of pamoic acid, a 9 micromolar pol beta inhibitor, and found that it binds in a single pocket at the surface of the 8 kDa domain of pol beta. However, docking studies provided five possible conformations for pamoic acid in this site. NMR experiments were performed on the complex to select a single conformation among the five retained. Chemical Shift Mapping data confirmed pamoic acid binding site found by docking while NOESY and saturation transfer experiments provided distances between pairs of protons from the pamoic acid and those of the 8 kDa domain that allowed the identification of the correct conformation.ConclusionCombining NMR experiments on the complex with docking results allowed us to build a three-dimensional structural model. This model serves as the starting point for further structural studies aimed at improving the affinity of pamoic acid for binding to DNA polymerase beta.