Xianzhong Xu

Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone.

Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the females antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBP-mediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP·pheromone complex. Although CquiOBP1 binds MOP in a pH-dependent fashion, it lacks the C terminus required for the pH-dependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

Background The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. Methodology By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. Conclusion We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

NMR Structure of Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1): Implications for pH-Sensitive Pheromone Detection,

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from A. transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multiangle light scattering, and (15)N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven alpha-helices (alpha1, L8-L23; alpha2, D27-F36; alpha3, R46-V62; alpha4, A73-M78; alpha5, D84-S100; alpha6, R107-L125; alpha7, M131-E141) that adopt an overall main-chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108, and C97/C117 and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141, and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (alpha7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139, and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, and V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions.

Reverse Micelle Encapsulation of Membrane-Anchored Proteins for Solution NMR Studies

Perhaps 5%-10% of proteins bind to membranes via a covalently attached lipid. Posttranslational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca(2+) signaling pathway in vision, and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol-4,5-bisphosphate. Both proteins have been successfully encapsulated in the lipid-extruded state and high-resolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.

Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding.

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.

Conformational dynamics of recoverin's Ca2+‐myristoyl switch probed by 15N NMR relaxation dispersion and chemical shift analysis

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca2+‐induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca2+‐myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on 15N‐labeled recoverin to probe main chain conformational dynamics. 15N NMR relaxation data suggest that Ca2+‐free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca2+ levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca2+‐dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca2+ saturated extruded state (R): T ↔ I ↔ R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τexδω < 1). The final step (I ↔ R) is much slower (τexδω > 1). The main chain structure of I is similar in part to the structure of half‐saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ↔ I may transiently increase the exposure of Ca2+‐binding sites to initiate Ca2+ binding that drives extrusion of the myristoyl group during I ↔ R. Proteins 2011;

Double electron-electron resonance probes Ca2+-induced conformational changes and dimerization of recoverin

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²⁺-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²⁺-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²⁺-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²⁺-free recoverin and 3.7 ± 0.1 nm for Ca²⁺-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²⁺-bound recoverin may represent an intermolecular distance between C39 and N120. ¹⁵N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²⁺-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca²⁺-free recoverin is monomeric. We propose that Ca²⁺-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²⁺-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²⁺-induced dimerization is functionally important but not well understood.

1H, 15N, and 13C chemical shift assignments of the mosquito odorant binding protein-1 (CquiOBP1) bound to the mosquito oviposition pheromone

An odorant-binding protein from the Southern house mosquito, Culex pipiens quinquefasciatus (Cqui-OBP1) binds to the mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide to facilitate the transport of MOP to membrane-bound odorant receptors. We report complete NMR chemical shift assignments of Cqui-OBP1 bound to the MOP pheromone obtained at pH 7.0 and 25°C (BMRB no. 16175).

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei.

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF‐hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF‐hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N‐terminal myristoyl group may be important for membrane binding. Non‐conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.

1 H, 15 N, and 13 C chemical shift assignments of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of Trypanosoma brucei. (BMRB no. 18011).