Barbara Seaton, Ph.D.

Research

Structure & Function of Proteins

The general theme underlying my research program involves the structure and function of proteins at the membrane. Most of our focus has been on peripheral membrane proteins, which act at the membrane surface. These include annexins, vitamin K-dependent blood coagulation proteins (e.g. prothrombin), lung surfactant proteins (e.g. SP-A), and phospholipase D. Functionally, these proteins are diverse and influence a wide range of physiological processes, e.g.: blood coagulation, membrane aggregation, cell fusion, host defense, and cell signalling. Mechanistically, these proteins have several common features, including the utilization of calcium ions for activation and/or membrane attachment, and specific interactions with phospholipids.

To gain the most detailed picture of these proteins and their behavior at the membrane interface, we use a combined approach. X-ray crystallography is central to our studies, and its high-resolution information is complemented by site-directed mutagenesis, vesicle binding studies, and various other biophysical or spectroscopic techniques, depending upon the project. Though the structure/function investigation of peripheral membrane proteins remains the primary focus in our lab, more recently we also have initiated studies on proteins involved in antibiotic resistance and transmembrane proteins.

The following studies are two recent examples of completed work and are representative of current work in the lab.

This work is supported primarily by NIH (GMS and HBL Institutes).

Annexin V self-assembly process on phospholipid membranes
may be mediated by cell surface proteoglycan heparan sulfate.

Annexin V, a protein with strong anticoagulant properties, is abundant in placenta and other tissues where it is believed to play a role in inhibiting thrombosis, or clot formation. The protein exhibits very high-affinity calcium-dependent binding interactions with acidic phospholipids (e.g. phosphatidylserine, PS), as well as specific binding to glycosaminoglycans (GAG) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. The annexin V monomer spontaneously forms trimers and extended trimer-based arrays on phospholipid membrane surfaces, and the membrane-bound annexin layer has been proposed to form a protective anti-thrombotic shield at these sites. Disruption of this shield is associated with certain annexinopathies (diseases related to annexin dysfunction) such as antiphospholipid syndrome, leading to pathologies such as repeated miscarriage and excessive thrombosis. The physical properties of annexin V suggest that its action is regulated by membrane signalling processes. Extracellular expression of PS at the cell surface, a signal for the initiation of processes such as blood coagulation and apoptosis, creates binding sites for annexin V. The protein binds the membrane and spontaneously crystallizes on its surface, producing its biologically active species. In this way, annexin V becomes responsive to physiological signals that would recruit the protein to the membrane surface and promote annexin oligomerization and subsequent cell function. Mechanisms of annexin V function and behavior at the membrane surface are under investigation in our lab. Much of our previous and ongoing work is aimed at elucidating the behavior of annexin V at the membrane surface, specifically the calcium-dependent membrane binding property and the trimer-based array formation on membrane surfaces.

In a recent study summarized below, we began to look at annexin-GAG interactions, using a combined approach of x-ray crystallography, site-directed mutagenesis, and surface plasmon resonance (SPR) binding measurements. The crystal structure of annexin V complexed with two heparin tetrasaccharides was obtained (PDB link). From the combined data, we identified and characterized specific high-affinity heparin binding sites on the annexin molecule and proposed a novel functional model. In this model, the proteoglycan heparin sulfate assists in the localization of annexin V to the cell surface membrane and/or stabilizes the entire molecular assembly to promote anticoagulation. We are currently examining whole cell and in vivo models to test this hypothesis.

Annexin V-Heparin Interactions
Left figure: Crystal structure of annexin V complex with heparin tetrasaccharides (HTS-1 and HTS-2). Gold spheres are calcium ions, which also denote the membrane binding surface of the protein. Right figure: Model of annexin-heparin interactions at the membrane surface. The chains represent the polysaccharides of a proteoglycan. Heparin binding sites (designated 1,2, and 3) were identified by x-ray crystallography, site-directed mutagenesis, and surface plasmon resonance (SPR) binding measurements. Sites 1 and 3 correspond to HTS-2 and HTS-1, respectively, from the crystal structure. Site 2 is predicted from a heparin binding consensus sequence and confirmed by SPR analysis of mutants made in this region. View Reference

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Investigating the mechanism of annexin IV-mediated aggregation
of phospholipid membranes and its regulation by phosphorylation.

Annexin IV, a close structural homologue of annexin V, shares many of its properties but has a few major differences. Like V, annexin IV binds to acidic phospholipid membranes in the presence of calcium and forms trimer-based arrays on membrane surfaces. However, membrane-bound annexin IV also promotes vesicular aggregation, a property that underlies important processes such as cell fusion. This property distinguishes it from annexin V, which inhibits membrane aggregation. The pro-aggregation capability of annexin IV is localized to its N-terminal region, which contains a regulatory phosphorylation site. Phosphorylation of threonine 6 inhibits annexin IV-mediated vesicle aggregation. Similarly, introduction of a negative charge by mutating threonine 6 into aspartate results in a phenotype that mimics the phosphorylated form and lacks the ability to aggregate membranes. We embarked upon structure-function studies of this T6D mutant through a combined approach of x-ray crystallography, cryoEM (in collaboration with Alain Brisson), and vesicle binding assays to discover how the N-terminus influences membrane aggregation in annexin IV. Our studies led us to the hypothesis that the N-terminus of annexin IV represents a conformational switch that can reversibly promote or inhibit vesicle aggregation.

The crystal structure of wild-type annexin IV (PDB code # 1ANN) depicts the N-terminus in an extended conformation, lying in a shallow cleft on the protein surface facing away from the membrane. The side chain of threonine 6 faces into the hydrophobic core of the protein. In contrast, the T6D annexin IV crystal structure (PDB link) shows that the N-terminus is released from the surface cleft to move freely in the solvent. A glycine and phenylalanine at positions 12 and 13 act as a hinge and an anchor, respectively, from which the preceding N-terminal strand separates from the rest of the protein. The dislocated N-terminus becomes so mobile in the T6D mutant that little of it is visible in the crystal structure (see figure below). This alteration obviously greatly affects the protein surface that includes the N-terminus. But how does the N-terminal conformational change mediate membrane aggregation? Together, the crystallographic and cryoEM results suggest the following mechanism. Two apposing membranes coated with annexin IV adhere to each other through homotropic annexin-annexin interactions on the protein surfaces containing the N-terminal region. Phosphorylation disrupts that surface by causing the release of the N-terminal strand, as observed for T6D. These results are shown below.

Crystal structures of wild-type (left) and T6D (right) annexin V, showing the N-terminal regions. The IIIAB loop and two calcium ions (black spheres) are features of the membrane-binding surface of annexin IV. View Reference

CryoEM micrographs of vesicles coated with (a) wild-type annexin IV or (b) T6D mutant annexin IV. Vesicle aggregation is observed in (a) but not (b). In (c), aggregated vesicles form junctions, as indicated by the arrow, in which four "stripes" of measurable thickness. The junctions correspond to two layers of annexin and two of lipid bilayer. This structure is modeled in (d).

Publications:

Pertinent References:

I. Capila, M.J. Hernaiz, Y.D. Mo, T.R. Mealy, B. Campos, J.R. Dedman, R.J. Linhardt, and B.A. Seaton (2001) "Annexin-V Heparin Oligosaccharide Complex Suggests Heparan Sulfate-Mediated Assembly on Cell Surfaces." Structure 9, Abstract

I. Capila, V.A. NanderNoot, T.R. Mealy, B.A. Seaton, and R.J. Linhardt (1999) "Interaction of heparin with annexin V." FEBS Lett. 446, 327-330. Abstract

M.A. Katezel, Y.D. Mo, T.R. Mealy, B. Campos, W. Bergsma-Schutter, A. Brisson, J.R. Dedman, and B.A. Seaton (2001) "Phosphorylation Mutants Elucidate the Mechanism of Annexin IV-Mediated Membrane Aggregation." Biochemistry 40, Abstract

Other Representative Publications: 

Concha, N.O., Head, J.F., Kaetzel, M.A., Dedman, J.R. and Seaton, B.A. (1992) Annexin V forms a calcium-dependent trimeric unit structure on phospholipid vesicles. FEBS. Lett. 314,159-162. Abstract

Concha, N.O., Head, J.F., Kaetzel, M.A., Dedman, J.R. and Seaton, B.A. (1993) Rat Annexin V Crystal Structure: CA2+-induced Conformational Changes. Science 261. 1321-1324. Abstract

Swairjo, M.A., Roberts. M.F., Campos, M.B., Dedman, J.R. and Seaton, B.A. (1994) 31P- and 1H-NMR Studies of the Interaction of Annexin V with Small Phosphatidic Acid-Containing Unilamellar Vesicles. Biochemistry 33, 10944-10950. Abstract

Swairjo, M.A., Concha, N.O., Kaetzel, M.A., Dedman, J.R. and Seaton, B.A. (1995) Ca2+ -bridging Mechanism and Phospholipid Head Group Recognition in the Membrane-binding Protein Annexin V. Nature Structural Biology 2, 968-974. Abstract

Jin, L., Seaton, B.A. and Head, J.F. (1997) Crystal Structure at 2.8Å Resolution of anabolic ornithine transcarbamylase from Escherichia coli. Nat. Struct. Biol. 4, 622-625. Abstract

Campos, M.B., Mo, Y.D., Mealy, T.R., Li, C.W., Swairjo, M.B., Balch, C., Head, J.F., Retzinger, G., Dedman, J.R. and Seaton, B.A. (1998) Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components. Biochemistry 37, 8004-8010. Abstract

Wu, F., Flach, C.R., Mealy, T.R., Seaton, B.A. and Mendelsohn, R. (1998) Domain structure and molecular conformation in annexin V/1,2-dimyristoyl-sn-glycero-3-phosphate/Ca2+ aqueous monolayers: a Brewster angle microscopy/infrared reflection-absorption spectroscopy study. Biophys J. 74, 3273-81. Abstract

Wu, F., Flach, C.R., Mealy, T.R., Seaton, B.A. and Mendelsohn, R. (1999) Stability of annexin V in ternary complexes with Ca2+ and anionic phospholipids: IR studies of monolayer and bulk phases. Biochemistry 38, 792-799. Abstract

Stieglitz, K., Seaton, B. and Roberts, M.F. (1999) The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid. J. Biol. Chem. 274, 35367-35374. Abstract

Stieglitz, K., Seaton, B. and Roberts, M.F. (2001) Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion. Biochemistry 40, 13954-63. Abstract

Alekshun, M.N., Levy, S.B., Mealy, T.R., Seaton, B.A. and Head, J.F. (2001) The crystal structure of MarR, a regulator of multiple antibiotic resistance, at 2.3A resolution. Nat Struct Biol. 8, 710-4. Abstract

Mo, Y., Campos, B., Mealy, T.R., Commodore, L., Head, J.F., Dedman, J.R., Seaton, B.A. (2003) Interfacial basic cluster in annexin V couples phospholipid binding and trimer formation on membrane surfaces. J. Biol. Chem. 278, 2437-43.

Selected Reviews:

Swairjo, M.A. and Seaton, B.A. (1994) Annexin Structure and Membrane Interactions: A Molecular Perspective. Annu. Rev. Biophys. Biomolec. Struct. 23, 193-213. Abstract

Seaton, B.A. editor. Annexins: Molecular Structure to Cellular Function (1996) R.G. Landes Company, Austin.

Seaton, B.A. and Roberts, M.F. "Peripheral Membrane Proteins" Structure, Function and Dynamics of Membranes (1996). K.Merz, Jr. and B. Roux, Eds. Birkhaüser, Springer-Verlag, New York, pp.355-403.

Seaton, B.A. and Dedman, J.R. (1998) Annexins. Biometals 11, 399-404. Abstract

Contact Us

Department of Physiology and Biophysics
Boston University School of Medicine
715 Albany Street
Boston MA 02118-2526

Phone:(617) 638-5061
Fax: (617) 638-4273
e-mail: seaton@medxtal.bu.edu