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author:("leroux, Annie")
1.  The Structure of Xis reveals the basis for Filament Formation and insight into DNA bending within a mycobacteriophage Intasome 
Journal of molecular biology  2013;426(2):412-422.
The Recombination Directionality Factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of Mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament, and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head-to-tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.
doi:10.1016/j.jmb.2013.10.002
PMCID: PMC3902635  PMID: 24112940
DNA recombination; mycobacteriophage Pukovnik; Xis; DNA bending; filament; structure
2.  Structure of a Highly Conserved Domain of Rock1 Required for Shroom-Mediated Regulation of Cell Morphology 
PLoS ONE  2013;8(12):e81075.
Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.
doi:10.1371/journal.pone.0081075
PMCID: PMC3857177  PMID: 24349032
3.  Structural Reorganization Triggered by Charging of Lys Residues in the Hydrophobic Interior of a Protein 
Structure(London, England:1993)  2012;20(6):1071-1085.
Summary
Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied the ionization of an internal Lys unfolded the protein globally. In 5 cases the internal charge triggered localized changes in structure and dynamics, and in 3 cases they promoted partial or local unfolding. Remarkably, in 15 proteins the ionization of the internal Lys resulted in no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pKa calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.
doi:10.1016/j.str.2012.03.023
PMCID: PMC3373022  PMID: 22632835
electrostatics; proteins; pKa values; buried charged NMR spectroscopy; staphylococcal nuclease
4.  The 1.75 Å resolution structure of fission protein Fis1 from Saccharomyces cerevisiae reveals elusive interactions of the autoinhibitory domain 
A 1.75 Å resolution crystal structure of the Fis1 cytoplasmic domain from Saccharomyces cerevisiae is reported which adopts a tetratricopeptide-repeat fold.
Fis1 mediates mitochondrial and peroxisomal fission. It is tail-anchored to these organelles by a transmembrane domain, exposing a soluble cytoplasmic domain. Previous studies suggested that Fis1 is autoinhibited by its N-terminal region. Here, a 1.75 Å resolution crystal structure of the Fis1 cytoplasmic domain from Saccharomyces cerevisiae is reported which adopts a tetratricopeptide-repeat fold. It is observed that this fold creates a concave surface important for fission, but is sterically occluded by its N-terminal region. Thus, this structure provides a physical basis for autoinhibition and allows a detailed examination of the interactions that stabilize the inhibited state of this molecule.
doi:10.1107/S1744309111029368
PMCID: PMC3212442  PMID: 22102223
tetratricopeptide repeats; protein–protein interactions; tail-anchoring; mitochondria; peroxisomes; membrane dynamics
5.  Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase 
Journal of the American Chemical Society  2011;133(35):14017-14026.
The hydrophobic effect—the free-energetically favorable association of non-polar solutes in water—makes a dominant contribution to binding of many systems of ligands and proteins. The objective of this study was to examine the hydrophobic effect in biomolecular recognition using two chemically different, but structurally similar hydrophobic groups—aliphatic hydrocarbons and aliphatic fluorocarbons—and to determine whether the hydrophobicity of the two groups could be distinguished by thermodynamic and biostructural analysis. This paper uses isothermal titration calorimetry (ITC) to examine the thermodynamics of binding of benzenesulfonamides substituted in the para position with alkyl and fluoroalkyl chains (H2NSO2C6H4-CONHCH2(CX2)nCX3, n = 0–4, X = H, F) to human carbonic anhydrase II (HCA II). Both alkyl and fluoroalkyl substituents contribute favorably to the enthalpy and the entropy of binding; these contributions increase as the length of chain of the hydrophobic substituent increases. Crystallography of the protein-ligand complexes indicates that the benzenesulfonamide groups of all ligands examined bind with similar geometry, that the tail groups associate with the hydrophobic wall of HCA II (which is made up of the side chains of residues Phe131, Val135, Pro202, and Leu204), and that the structure of the protein is indistinguishable for all but one of the complexes (the longest member of the fluoroalkyl series). Analysis of the thermodynamics of binding as a function of structure is compatible with the hypothesis that hydrophobic binding of both alkyl and fluoroalkyl chains to hydrophobic surface of carbonic anhydrase is due primarily to the release of non-optimally hydrogen-bonded water molecules that hydrate the binding cavity (including the hydrophobic wall) of HCA II and to the release of water molecules that surround the hydrophobic chain of the ligands. This study defines the balance of enthalpic and entropic contributions to the hydrophobic effect in this representative system of protein and ligand: hydrophobic interactions, here, seem to comprise approximately equal contributions from enthalpy (plausibly from strengthening networks among molecules of water hydrogen bonds) and entropy (from release of water from configurationally restricted positions).
doi:10.1021/ja2045293
PMCID: PMC3171206  PMID: 21790183
6.  Structure of Shroom domain 2 reveals a three-segmented coiled-coil required for dimerization, Rock binding, and apical constriction 
Molecular Biology of the Cell  2012;23(11):2131-2142.
The Shrm SD2 region contains a core that adopts a novel three-segmented dimer required for Rock binding. Conserved interfaces critical for Rock binding, ppMLC levels, and the formation of contractile cytoskeletal networks are identified. The complex is likely tetrameric, which suggests that conformational changes within SD2 are likely upon Rock binding.
Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho­logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm–Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm–Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock–Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm–Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.
doi:10.1091/mbc.E11-11-0937
PMCID: PMC3364177  PMID: 22493320
7.  A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC 
A TCR derived from a patient with relapsing-remitting multiple sclerosis engages the self-peptide myelin basic protein in the context of HLA-DQ1 in a very unusual way.
Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide–major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-α chain with the MHC class II β chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3β loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3α loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.
doi:10.1084/jem.20100725
PMCID: PMC3023130  PMID: 21199956
8.  The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways▿  
Journal of Virology  2010;84(19):9995-10003.
The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01_AE (AE) strain is seen principally in Southeast Asia. AE protease differs by ∼10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.
doi:10.1128/JVI.00505-10
PMCID: PMC2937823  PMID: 20660190
9.  Preliminary crystallographic studies of the regulatory domain of response regulator YycF from an essential two-component signal transduction system 
The regulatory domain of response regulator YycF from an essential two-component signal transduction system has been crystallized and X-ray data have been collected at 1.95 Å resolution.
YycGF is a crucial signal transduction system for the regulation of cell-wall metabolism in low-G+C Gram-positive bacteria, which include many important human pathogens. The response regulator YycF receives signals from its cognate histidine kinase YycG through a phosphotransfer reaction and elicits responses through regulation of gene expression. The N-terminal regulatory domain of YycF from Bacillus subtilis was overproduced and purified. The protein was crystallized and X-ray data were collected to 1.95 Å resolution with a completeness of 97.7% and an overall R merge of 7.7%. The crystals belonged to space group P3121, with unit-cell parameters a = b = 59.50, c = 79.06 Å.
doi:10.1107/S1744309109022696
PMCID: PMC2705644  PMID: 19574649
response regulators; two-component systems; signal transduction; Gram-positive bacteria; YycF; YycG; transcription factor; cell wall
10.  Structure and Mechanistic Implications of a Uroporphyrinogen III Synthase–Product Complex†,‡ 
Biochemistry  2008;47(33):8648-8655.
Uroporphyrinogen III synthase (U3S) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen III (uro’gen III) isomer during heme biosynthesis. Here, we report four apoenzyme and one product complex crystal structures of the Thermus thermophilus (HB27) U3S protein. The overlay of eight crystallographically unique U3S molecules reveals a huge range of conformational flexibility, including a “closed” product complex. The product, uro’gen III, binds between the two domains and is held in place by a network of hydrogen bonds between the product’s side chain carboxylates and the protein’s main chain amides. Interactions of the product A and B ring carboxylate side chains with both structural domains of U3S appear to dictate the relative orientation of the domains in the closed enzyme conformation and likely remain intact during catalysis. The product C and D rings are less constrained in the structure, consistent with the conformational changes required for the catalytic cyclization with inversion of D ring orientation. A conserved tyrosine residue is potentially positioned to facilitate loss of a hydroxyl from the substrate to initiate the catalytic reaction.
doi:10.1021/bi800635y
PMCID: PMC2852885  PMID: 18651750
11.  Biochemical and Structural Studies of Yeast Vps4 Oligomerization 
Journal of molecular biology  2008;384(4):878-895.
The ESCRT pathway functions in vesicle formation at the multivesicular body, the budding of enveloped RNA viruses such as HIV-1, and the final abscission stage of cytokinesis. As the only known enzyme in the ESCRT pathway, the AAA ATPase Vps4 provides the energy required for multiple rounds of vesicle formation. Like other Vps4 proteins, yeast Vps4 cycles through two states: a catalytically inactive disassembled state that we show here is a dimer, and a catalytically active higher order assembly that we have modeled as a dodecamer composed of two stacked hexameric rings. We also report crystal structures of yeast Vps4 proteins in the apo- and ATPγS-bound states. In both cases, Vps4 subunits assembled into continuous helices with six-fold screw axes that are analogous to helices seen previously in other Vps4 crystal forms. The helices are stabilized by extensive interactions between the large and small AAA ATPase domains of adjacent Vps4 subunits, suggesting that these contact surfaces may be used to build both the catalytically active dodecamer and catalytically inactive dimer. Consistent with this model, we have identified interface mutants that specifically inhibit Vps4 dimerization, dodecamerization, or both. Thus, the Vps4 dimer and dodecamer likely form distinct but overlapping interfaces. Finally, our structural studies have allowed us to model the conformation of a conserved loop (Pore Loop 2) that is predicted to form an arginine-rich pore at the center of one of the Vps4 hexameric rings. Our mutational analyses demonstrate that Pore Loop 2 residues Arg241 and Arg251 are required for efficient HIV-1 budding, thereby supporting a role for this “arginine collar” in Vps4 function.
doi:10.1016/j.jmb.2008.09.066
PMCID: PMC2632936  PMID: 18929572
Vps4; AAA ATPase; Oligomerization; MVB pathway; X-ray Crystallography
12.  Autoregulation of the Rsc4 Tandem Bromodomain by Gcn5 Acetylation 
Molecular cell  2007;27(5):817-828.
SUMMARY
An important issue for chromatin remodeling complexes is how their bromodomains recognize particular acetylated lysine residues in histones. The Rsc4 subunit of the yeast remodeler RSC contains an essential tandem bromodomain (TBD) that binds acetylated K14 of histone H3 (H3K14ac). We report a series of crystal structures that reveal a compact TBD that binds H3K14ac in the second bromodomain and, remarkably, binds acetylated K25 of Rsc4 itself in the first bromodomain. Endogenous Rsc4 is acetylated only at K25, and Gcn5 is identified as necessary and sufficient for Rsc4 K25 acetylation in vivo and in vitro. Rsc4 K25 acetylation inhibits binding to H3K14ac, and mutation of Rsc4 K25 results in altered growth rates. These data suggest an autoregulatory mechanism in which Gcn5 performs both the activating (H3K14ac) and inhibitory (Rsc4 K25ac) modifications, perhaps to provide temporal regulation. Additional regulatory mechanisms are indicated as H3S10 phosphorylation inhibits Rsc4 binding to H3K14ac peptides.
doi:10.1016/j.molcel.2007.08.018
PMCID: PMC2788556  PMID: 17803945
13.  Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor 
The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.
doi:10.1107/S1744309108027048
PMCID: PMC2564891  PMID: 18931427
14.  Structure of a 6-pyruvoyltetrahydropterin synthase homolog from Streptomyces coelicolor  
The X-ray crystal structure of a 6-pyruvoyltetrahydropterin synthase homolog of unknown function has been determined at 1.5 Å resolution. The protein retains residues required for pterin binding, but nearly all catalytic residues are missing.
The X-ray crystal structure of the 6-pyruvoyltetrahydropterin synthase (PTPS) homolog from Streptomyces coelicolor, SCO 6650, was solved at 1.5 Å resolution. SCO 6650 forms a hexameric T-fold that closely resembles other PTPS proteins. The biological activity of SCO 6650 is unknown, but it lacks both a required active-site zinc metal ion and the essential catalytic triad and does not catalyze the PTPS reaction. However, SCO 6650 maintains active-site residues consistent with binding a pterin-like substrate.
doi:10.1107/S1744309108027048
PMCID: PMC2564891  PMID: 18931427
SCO 6650; Streptomyces coelicolor; 6-pyruvoyltetrahydropterin synthase homolog
15.  CD94-NKG2A recognition of human leukocyte antigen (HLA)-E bound to an HLA class I leader sequence 
The recognition of human leukocyte antigen (HLA)-E by the heterodimeric CD94-NKG2 natural killer (NK) receptor family is a central innate mechanism by which NK cells monitor the expression of other HLA molecules, yet the structural basis of this highly specific interaction is unclear. Here, we describe the crystal structure of CD94-NKG2A in complex with HLA-E bound to a peptide derived from the leader sequence of HLA-G. The CD94 subunit dominated the interaction with HLA-E, whereas the NKG2A subunit was more peripheral to the interface. Moreover, the invariant CD94 subunit dominated the peptide-mediated contacts, albeit with poor surface and chemical complementarity. This unusual binding mode was consistent with mutagenesis data at the CD94-NKG2A–HLA-E interface. There were few conformational changes in either CD94-NKG2A or HLA-E upon ligation, and such a “lock and key” interaction is typical of innate receptor–ligand interactions. Nevertheless, the structure also provided insight into how this interaction can be modulated by subtle changes in the peptide ligand or by the pairing of CD94 with other members of the NKG2 family. Differences in the docking strategies used by the NKG2D and CD94-NKG2A receptors provided a basis for understanding the promiscuous nature of ligand recognition by NKG2D compared with the fidelity of the CD94-NKG2 receptors.
doi:10.1084/jem.20072525
PMCID: PMC2275392  PMID: 18332182

Results 1-15 (15)