C. James McKnight, Ph.D.
Associate Professor of Physiology and Biophysics
B.S. Washington College
Ph.D. UT Southwestern Medical Center
I. Actin-Binding by Headpiece Motifs
One focus in the lab centers on the intriguing, modular F-actin binding “headpiece” motif. The headpiece motif is a compact (~70 amino acid) domain localized at the extreme C-terminus of much larger “core” domains from several functionally diverse classes of actin-binding proteins. These headpiece-containing proteins include villin, supervillin, dematin, limatin and TalB, and their functions range from maintenance of the cytoskeleton and cell-cell adherens junctions, to potential roles in development. We are interested in determining the three dimensional structures of headpiece motifs and detailing their interaction with actin, their core domains, and regulatory kinases.
II. A Minimalist Folded Protein
Another area of interest is the development of minimal length folded proteins to bridge the gap between experimental and computational approaches to protein folding. We have shown that headpiece domain of villin contains a “subdomain” of only 35 residues that folds to form a novel three helix structure. The villin subdomain is one of the shortest amino acid sequences to fold to a monomeric native state in the absence of disulfide bonds or bound metals/ligands. We are using a mutational approach to address the question of how this short sequence encodes the information for a fully folded protein.
III. Modeling Low Density Lipoprotein (LDL) Assembly & Secretion
LDL is the major cholesterol transporting lipoprotein, and high LDL levels are correleated with Arthelroscrosis. We are investigating the early events in the formation of LDL and VLDL particles within the cell. We have modelled the first 17% of ApoB (ApoB-17) onto the crystal structure lipovitellin, whose sequence is homologous the N-terminal region of ApoB. We then used our ApoB-17 model to dock to a model lipid emulsion particle. This is one possible model for nascent LDL particle formation. We have tested the lipovitellin-based model by limited proteolysis and have created a bank of constructs corresponding to the individual domains for further structural and biophysical study.
Brown, J.M., Vardar-Ulu, D., McKnight, C.J. (2009) “How to arm a supervillin: Designing F-actin binding activity into supervillin headpiece,” J. Mol. Biol., 393, 608-618.
Grasko, J.M., Hooper, A.J., Brown, J.W., McKnight, C.J. & Burnett, J.R. (2009) “A novel missense HGD gene mutation, K57N, in a patient with alkaptonuria,” Clinica Chimica Acta, 403, 254-6.
Meng, J. & McKnight, C.J. (2009) “Heterogeneity and dynamics in villin headpiece crystal structures,” Acta Cryst. D, 65, 470-6.
Chen, L., Jiang, Z.G., Khan, A.A., Chishti, A.H. & McKnight, C.J. (2009) “Dematin exhibits a natively unfolded core domain and an independently folded headpiece domain,” Protein Science, 18, 629-636.
Mitsche, M., Wang, L., Jiang, Z.G., McKnight, C.J. & Small, D.M. (2009) “Interfacial properties of a complex multi-domain 490 amino acid peptide derived from apolipoprotein B (residues 292-782),” Langmir, 25, 2322-2330.
Vugmeyster, L. & McKnight, C.J. (2008) “Phosphorylation-Induced Changes in backbone dynamics of the dematin headpiece c-terminal domain,” J. Biomolec. NMR, 43, 39-50.
Vugmeyster, L. & McKnight, C.J. (2008) “Slow motions in chicken villin headpiece subdomain probed by cross-correlated NMR relaxation of amide NH bonds in successive residues,” Biophysical J., 95. 5941-50.
Jiang, Z.G, Liu, Y., Hussain, M.M., Atkinson, D. & McKnight, C.J. (2008) “Reconstituting initial events during the assembly of ApoB-containing lipoproteins in a cell-free system,” J. Mol. Biol., 383, 1181-1194.
Meng, J. & McKnight, C.J. (2008) “Crystal structure of a pH-stabilized mutant of villin headpiece,” Biochemistry, 47, 4644-4650.
Meng, J., Parroche, P., Golenbock, D.T. & McKnight, C.J. (2008) “The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14,” J. Biol. Chem., 283, 3376-3384.
Burnett, J.R., Zhong, S., Jiang, Z.G., Hooper, A.J., Fisher, E.A., McLeod, R.S., Zhao, Y., Barrett, H.R., Hegele, R.A., van Bockxmeer, F.M., Zhang, H., Vance, D.E., McKnight, C.J., & Yao, Z. (2007) “Missense mutations in ApoB within the βa1 domain of human ApoB-100 result in impaired secretion of apoB and ApoB-containing lipoproteins in familial hypobetalipoproteinemia,” J. Biol. Chem., 282, 24770-24283.
Smirnov, S.L., Isern, N.G., Jiang, Z.G., Hoyt, D.W. & McKnight, C.J. (2007) “The isolated sixth gelsolin repeat and headpiece domain of villin bundle F-actin in the presence of calcium and are linked by a 40-residue unstructured sequence,” Biochemistry, 46, 7488-7496.
Latz, E., Verma, A., Visintin,A., Gong, M., Sirois, C.M., Klein, D., Monks, B.G., McKnight, C.J., Duprex, W.P., Espevik, T., & Golenbock, D.T. (2007) “Ligand induced conformational changes in TLR9 result in allostereic receptor activation,” Nature Immunology, 8, 772-779.
Jiang, Z.G., Simon, M.N., Wall, J.S., & McKnight, C.J., (2007) “Structural analysis of reconstituted lipoproteins containing the N-terminal domain of apolipoprotein B,” Biophysical J., 92, 4097-4108.
Jiang, Z.G., Gantz, D., Bullitt, E., & McKnight, C.J., (2006) “Defining lipid interacting domains in the N-terminal region of apolipoprotein B, Biochemistry,” 45; 11799-11808.
Jiang, Z.G. & McKnight, C.J. (2006) “A phosphorylation induced change in dematin headpiece,” Structure, 14, 379-387.
Grey M.J., Tang Y., Alexov E., McKnight C.J., Raleigh D.P., & Palmer AG III. (2006) “Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: Contribution of His41 to the pH-dependent stability of the N-terminal subdomain,” J. Mol Biol., 355, 1078-1094.
Tang Y., Grey M.J., McKnight J., Palmer A.G. III, & Raleigh D.P. (2006) “Multistate folding of the villin headpiece domain,” J. Mol. Biol., 355, 1066-1077.
Department of Physiology and Biophysics
Boston University School of Medicine
700 Albany Street
Boston MA 02118-2526
Phone: (617) 638-4042
Fax: (617) 638-4041