Barbara A. Seaton

The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3 A resolution.

MarR is a regulator of multiple antibiotic resistance in Escherichia coli. It is the prototypical member of the MarR family of regulatory proteins found in bacteria and archaea that play important roles in the development of antibiotic resistance, a global health problem. Here we describe the crystal structure of the MarR protein, determined at a resolution of 2.3 Å. This is the first reported crystal structure of a member of this newly-described protein family. The structure shows MarR as a dimer with each subunit containing a winged-helix DNA binding motif.

Structural basis of membrane binding by Gla domains of vitamin K-dependent proteins.

In a calcium-dependent interaction critical for blood coagulation, vitamin K–dependent blood coagulation proteins bind cell membranes containing phosphatidylserine via γ-carboxyglutamic acid–rich (Gla) domains. Gla domain–mediated protein-membrane interaction is required for generation of thrombin, the terminal enzyme in the coagulation cascade, on a physiologic time scale. We determined by X-ray crystallography and NMR spectroscopy the lysophosphatidylserine-binding site in the bovine prothrombin Gla domain. The serine head group binds Gla domain–bound calcium ions and Gla residues 17 and 21, fixed elements of the Gla domain fold, predicting the structural basis for phosphatidylserine specificity among Gla domains. Gla domains provide a unique mechanism for protein-phospholipid membrane interaction. Increasingly Gla domains are being identified in proteins unrelated to blood coagulation. Thus, this membrane-binding mechanism may be important in other physiologic processes.

Annexin V–Heparin Oligosaccharide Complex Suggests Heparan Sulfate–Mediated Assembly on Cell Surfaces

BACKGROUND Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.

Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif

Lactadherin is a phosphatidyl-l-serine (Ptd-l-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-l-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-l-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7Å resolution. The bovine Lact-C2 structure has a β-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the β-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-l-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Annexin V forms calcium-dependent trimeric units on phospholipid vesicles

The quaternary structure of annexin V, a calcium‐dependent phospholipid binding protein, was investigated by chemical cross‐linking. Calcium was found to induce the formation of trimers, hexamers, and higher aggregates only when anionic phospholipids were present. Oligomerization occurred under the same conditions as annexin—vesicle binding. A model is proposed in when cell stimulation leads to calcium‐induced organization of arrays of annexin V lining the inner membrane surface, thus altering properties such as permeability and fluidity.

Review: Structural determinants of pattern recognition by lung collectins.

Host defense roles for the lung collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), were first suspected in the 1980s when molecular characterization revealed their sequence homology to the acute phase reactant of serum, mannose-binding lectin. Surfactant protein A and SP-D have since been shown to play diverse and important roles in innate immunity and pulmonary homeostasis. Their location in surfactant ideally positions them to interact with air-space pathogens. Despite extensive structural similarity, the two proteins show many functional differences and considerable divergence in their interactions with microbial surface components, surfactant lipids, and other ligands. Recent crystallographic studies have provided many new insights relating to these observed differences. Although both proteins can participate in calcium-dependent interactions with sugars and other polyols, they display significant differences in the spatial orientation, charge, and hydrophobicity of their binding surfaces. Surfactant protein D appears particularly adapted to interactions with complex carbohydrates and anionic phospholipids, such as phosphatidylinositol. By contrast, SP-A shows features consistent with its preference for lipid ligands, including lipid A and the major surfactant lipid, dipalmitoylphosphatidylcholine. Current research suggests that structural biology approaches will help to elucidate the molecular basis of pulmonary collectin—ligand recognition and facilitate development of new therapeutics based upon SP-A and SP-D.

Interaction of heparin with annexin V

The energetics and kinetics of the interaction of heparin with the Ca+2 and phospholipid binding protein annexin V, was examined and the minimum oligosaccharide sequence within heparin that binds annexin V was identified. Affinity chromatography studies confirmed the Ca+2 dependence of this binding interaction. Analysis of the data obtained from surface plasmon resonance afforded a K d of ∼21 nM for the interaction of annexin V with end‐chain immobilized heparin and a K d of ∼49 nM for the interaction with end‐chain immobilized heparan sulfate. Isothermal titration calorimetry showed the minimum annexin V binding oligosaccharide sequence within heparin corresponds to an octasaccharide sequence. The K d of a heparin octasaccharide binding to annexin V was ∼1 μM with a binding stoichiometry of 1:1.

Crystallographic analysis of calcium-dependent heparin binding to annexin A2.

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

Effect of vesicle composition and curvature on the dissociation of phosphatidic acid in small unilamellar vesicles - a 31P-NMR study

Sonicated small unilamellar vesicles (SUVs) containing phosphatidic acid (PA) give two PA 31P-NMR resonances corresponding to PA molecules in the inner and outer leaflets of the bilayer. This NMR differentiation between the two monolayers is not due to a pH gradient across the membrane but instead reflects differential packing in the inner and outer leaflets imposed by the highly curved SUV surface. The apparent pKa of the outer-leaflet PA increases with decreasing surface curvature and with increasing PA content. The estimated relationship between the apparent pKa of the outer-leaflet PA headgroup and vesicle curvature may provide a qualitative probe for effects related to surface curvature in these model-membrane systems.

The Role of Interfacial Binding in the Activation ofStreptomyces chromofuscus Phospholipase D by Phosphatidic Acid

The Streptomyces chromofuscusphospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH ≥ 7 in the presence of EGTA that is weakened as Ca2+ or Ba2+ are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba2+ was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA·Ca2+ surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca2+ modulation of PA·PLD binding involves protein aggregation that may be the critical interaction for activation.