Research Summary

A thread of actin filaments runs continuously through my research. My earliest publications are based on studies of the regulation of heart muscle contraction through the binding and release of calcium ions by the troponin/tropomyosin system. During my postdoctoral period, I learned to apply spectroscopic methods, particularly fluorescence, to the study of these systems. In my early years at UBC, I chemically modified actin and actin binding proteins with a variety of fluorescent reagents and probes to learn more about the structure and dynamics of these proteins in solution. On my first sabbatical (in Edmonton, with L. B. Smillie), I expanded the base of my studies to include non-muscle contractile and motile systems. This took me into the circulatory system, both intra and extracellularly, with platelets and blood plasma, respectively. Subsequent sabbaticals at Oxford (with D. I. Stuart and E. Y. Jones) and Uppsala (with R. C. Robinson) involved me in protein crystallographic determination of the molecular mechanism by which one particular actin binding protein, gelsolin, carries out its functions, both within cells and in extracellular fluids. This progression led to my involvement with the formation of the UBC Centre for Blood Research (http://www.cbr.ubc.ca), a recently established group of scientists with overlapping interests in circulatory research, but from different departments at UBC.

Gelsolin has been the main focus of my research for the past decade. I took gelsolin, prepared in my laboratory at UBC, with me to the Laboratory of Molecular Biophysics in Oxford in order to try to crystallize it. By then, my students and I had performed a number of chemical modifications on gelsolin in solution and felt we had a fairly good idea of how it functioned. I was able, with the help of a former M.Sc. student of mine, Robert Robinson, then a doctoral candidate in Oxford, to crystallize and solve the structure of the calcium-free, inactive form of gelsolin prepared from horse blood plasma. Since then, we have tried several different methods to obtain a crystallographic structure of calcium-bound, active gelsolin. Our successes to date have included solving the structure of the calcium-activated C-terminal half of gelsolin, both on its own and in a complex with actin. We learned that the activation process involves large movements of the domains of gelsolin relative to each other, generally with little refolding of the polypeptide chains within each domain. We also have solved the structure of a complex formed between actin and a fragment of gelsolin that consists of the entire first domain, G1, plus an additional stretch of polypeptide that normally constitutes the N-terminal portion of the second domain, G2. That G1 independently is able to bind actin was known, but we demonstrated that the following portion of polypeptide chain also binds tightly at the surface of actin. This could direct G2 toward a binding site that explains the role of that domain in making first contact between gelsolin and an actin filament, the first step in the filament severing activity of gelsolin.

We made two additional crystallographic advances during my latest sabbatical in Sweden. The first is that my student at UBC, Dunja Urosev, grew crystals of the N-terminal half of gelsolin bound to actin. Bob Robinson and I solved the structure, which produced several important results, now published in EMBO J. 1) All three of the gelsolin subunits share significant interfaces with the same actin monomer. 2) The position of the second domain, G2, of gelsolin on actin is consistent with what we expected from the structure of the complex of actin with the “G1-plus” fragment of gelsolin. G2 would be able to attach to two longitudinally arranged actin neighbours in the original Holmes model of an actin filament. Attachment to the second actin would be analogous to how G1 attaches to the first, but with a positively charged Arg residue on G2 replacing the positively charged Asp-to-Ca2+ pair on G1 that interacts with the negative Glu-167 side chain of the participating actin. 3) While the two halves of gelsolin in the absence of calcium appear very similar in form, once activated, they are distinctly different in appearance. These features permit modeling of the activation of gelsolin to start with release of the latches that render the actin-binding surfaces on the individual gelsolin domains inaccessible in the absence of calcium ions. Activated G2 can bind to two adjacent actin units along a filament. Interaction of the N-terminal portion of G2 with actin directs G1 to the junction between two actin units one step down the filament from the site of attachment of G2. Simultaneously, the second half of gelsolin, tethered by an extended polypeptide linker to the first, reaches around to position G4 near its binding site between two actin units across the filament from the G1 site. Binding of G1 and G4 effects severing of the actin filament, leaving one of the newly created ends (the one that normally would be faster growing) capped and unable to be extended. The results have direct implications for understanding the basis of the inherited disease, familial amyloidosis (Finnish type), and the “uncapping” activities of certain polyphosphoinositides on gelsolin-capped actin filaments.

The second advance referred to above is that I was able to grow crystals of a complex that gel electrophoresis shows to involve intact gelsolin with two actin molecules. This complex contains calcium ions and could provide the first direct experimental model of the structure of the capped end of a newly severed actin filament. Preliminary diffraction studies show these crystals to diffract, but all of the diffraction could be explained by a model that consists only of the N-terminal half of gelsolin bound to a single actin monomer. We have encountered the same result with our new crystals of complexes that involve one gelsolin molecule with three actins, and one gelsolin molecule with one actin. Effort is now being expended to alter and optimize the crystallization conditions to enhance diffraction quality and resolution. In addition, we are attempting to crystallize complexes that involve actin with a variety of cloned molecular fragments of gelsolin.

Technologies & Methods
Projects

We now are reaping the fruits of the work described above. We have complete x-ray diffraction data sets for solving the structures of several proteins and protein complexes, with more to come:

a) A novel structure of the horse G1-G3/actin complex, with G2 and G3 oriented differently on the actin surface than previously observed. The data will contribute to our understanding of how gelsolin is activated to bind to the side of an actin filament and subsequently sever that filament.

b) A novel crystal structure of native actin, grown in the presence of a chaperone protein, Hsp27.

c) The first structure of a complex formed between recombinant human gelsolin G1-G3 and actin. Proc. Natl. Acad. Sci. USA 106: 13713-13718 (2009)

d) The first structure of domain 6 of villin, a member of the gelsolin superfamily of proteins. J. Biol. Chem. 284: 21265-21269 (2009)

e) The first structure of the C-terminal half of adseverin, a tailless homolog of gelsolin. Proc. Natl. Acad. Sci. USA 106: 13719-13724 (2009)

f) The structure of a protein fragment comprising domains 2 and 3 of human capG, a member of the gelsolin superfamily of regulators of actin dynamics.

g) The structure of horse serum albumin, only the second serum albumin structure in the Protein Data Bank. The sequences of horse and human serum albumins differ by about a quarter, and we are analyzing the consequences of the differences.

Bio

Sabbatical leave, Uppsala (R.C. Robinson, 2002-2003).
Sabbatical leave, Oxford (E.Y. Jones and D. Stuart, 1994-95)
Sabbatical leave, Alberta (L.B. Smillie, 1983-84)
Postdoctoral, Weizmann Institute (I. Z. Steinberg, 1977-78)
Ph.D. (Biochemistry), University of Alberta, (C.M. Kay, 1973-77)
B.Sc., University of Manitoba, 1972

Publications
  1. Hui Wang, Robert C. Robinson and Leslie D. Burtnick, “The structure of native G-actin”, Cytoskeleton 67 , 456-465 (2010).
  2. *Nag, S., *Ma, Q., *Wang, H., Chumnarnsilpa, S., Lee, W.L., Larsson, M., Kannan, B., Hernandez-Valladares, M., Burtnick, L.D., and Robinson, R.C., “Ca2+-binding by domain 2 plays a critical role in the activation and stabilization of gelsolin.” Proc. Natl. Acad. Sci. (USA) 106: 13713-13718 (2009). (* co-“first authors”)
  3. Wang, H., Chumnarnsilpa, S., Loonchanta, A., Li., Q., Kwan, L., Robine, S., Larssen, M., Mihalek, I., Burtnick, L.D., and Robinson, R.C., “Helix straightening as an activation mechanism in the gelsolin superfamily of actin regulatory proteins”, J. Biol. Chem. 284: 21265-21269 (2009).
  4. Chumnarnsilpa, S., Lee, W.L., Nag, S., Kannan, B., Larsson, M., Burtnick, L.D., and Robinson, R.C., “The crystal structure of the C terminus of adseverein reveals the actin-binding interface.” Proc. Natl. Acad. Sci. (USA) 106: 13719-13724 (2009).
  5. Zaccai, N.R., May, A.P., Robinson, R.C., Burtnick, L.D., Crocker, P.R., Brossmer, R., Kelm, S., and Jones, E.Y. “Crystallographic and in silico Analysis of the Sialoside Binding Characteristics of the Siglec Sialoadhesin”, J. Mol. Biol. 365: 1469-1479 (2007).
  6. Chumnarnsilpa, S., Loonchanta, A., Xui, B., Choe, H., Urosev, D., Wang, H., Lindberg, U., Burtnick, L.D., and Robinson, R.C., “Calcium Ion Exchange in Crystalline Gelsolin”, J. Mol. Biol. 357: 773-782 (2006).
  7. Urosev, D., Ma, Q., Tan, A.L.C., Robinson, R.C., and Burtnick, L.D., “The Structure of Gelsolin Bound to ATP”, J. Mol. Biol. 357: 765-772 (2006).
  8. Aguda, A.H., Burtnick, L.D., and Robinson, R.C., “The State of the Filament”, EMBO Reports 6: 220-226 (2005).
  9. Irobi, E., Aguda, A.H., Larsson, M., Guerin, C., Yin, H.L., Burtnick, L.D., Blanchoin, L., and Robinson, R.C., “Structural Basis of Actin Sequestration by Thymosin-4: Implications for WH2 Proteins”, EMBO J. 23: 3599-3608 (2004).
  10. Burtnick, L.D., Urosev, D., Irobi, E., Narayan, K., and Robinson, R.C., “Structure of the N-terminal Half of Gelsolin Bound to Actin: Roles in Severing, Apoptosis and FAF”, EMBO J. 23: 2713-2722 (2004).
  11. Narayan, K., Chumnarnsilpa, S., Choe, H., Irobi, E., Urosev, D., Lindberg, U., Schutt, C.E., Burtnick, L.D., and Robinson, R.C., “Activation in isolation: Exposure of the actin-binding site in the C-terminal half of gelsolin does not require actin.”, FEBS Lett. 552: 82-85 (2003).
  12. Irobi, E., Burtnick, L.D., Urosev, D., Narayan, K., and Robinson, R.C., “From the first to the second domain of gelsolin: A common path on the surface of actin?” FEBS Lett. 552: 86-90 (2003).
  13. Choe, H., Burtnick, L.D., Mejillano, M., Yin, H., Robinson, R.C., and Choe, S., “The calcium activation of gelsolin: Insights from the 3 Å structure of the G4-G6/actin complex”, J. Mol. Biol. 324: 691-702 (2002).
  14. Robinson, R.C., Choe, S., and Burtnick, L.D., “The disintegration of a molecule: The role of gelsolin in FAF, familial amyloidosis (Finnish type) ”, Proc. Natl. Acad. Sci. (USA) 98: 2117-2118 (2001).
  15. Robinson, R.C., Mejillano, M., Le, V., Burtnick, L.D., Yin, H., and Choe, S., “Domain Movement in Gelsolin: A Calcium-activated Switch”, Science 286: 1939-1942 (1999).
  16. Robinson, R.C., Radziejewski, C., Spraggon, G., Greenwald, J., Kostura, M., Burtnick, L.D., Choe, S., Stuart, D.I., and Jones, E.Y., “The Structures of the Neurotrophin 4 homodimer and the Brain-Derived Neurotrophic Factor/Neurotrophin 4 Heterodimer Reveal a Common TrK-Binding Site”, Protein Science 8: 2589-2597 (1999).
  17. Koepf, E.K., Hewitt, J., Vo, H., MacGillivray, R.T.A., and Burtnick, L.D., “Equus caballus Gelsolin: cDNA sequence and Protein Structural Implications”, Eur. J. Biochem. 251: 613-621 (1998).
  18. Burtnick, L.D., Koepf, E.K., Grimes, J., Jones, E.Y., Stuart, D.I., McLaughlin, P.J., and Robinson, R.C., “The Crystal Structure of Plasma Gelsolin: Implications for Actin Severing, Capping and Nucleation”, Cell 90: 661-670 (1997).
  19. Koepf, E.K. and Burtnick, L.D., “Multiple Pathways for Denaturation of Horse Plasma Gelsolin”, Biochem. Cell Biol. 74: 101-107 (1996).
  20. Din, N., Forsythe, I.J., Burtnick, L.D., Gilkes, N.R., Miller, R.C., Warren, R.A.J., and Kilburn, D.G., “The Cellulose-Binding Domain of Endoglucanase A (CenA) from Cellulomonas fimi: Evidence for the Involvement of Tryptophan Residues in Binding”, Molecular Micro. 11: 747-755 (1994).
  21. Koepf, E.K., and Burtnick, L.D., “Horse Plasma Gelsolin Labelled with Fluorescein Isothiocyanate Responds to Calcium and Actin”, Eur. J. Biochem. 212: 713-718 (1993).
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