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.
Chen, L., Brown, J.W., Mok, Y.F., Hatters, D.M. & McKnight, C.J. (2013) The allosteric mechanism induced by protein kinase A (PKA) phosphorylation of dematin (band 4.9), J. Biol. Chem., 288, 8313-8320. PMID: 23355471; PMCID: PMC3605649.
Brown, J.W., Farelli, J.D. & McKnight, C.J. (2012) On the unyielding hydrophobic core of villin headpiece, Protein Sci., 21, 647-654. PMID: 22467489.
Li, X., Marinkovic, M., Russo, C., McKnight, C.J., Coetzer, T.L. & Chishti, A.H. (2012) Identification of a specific region of Plasmodium falciparum EBL-1 that binds to host receptor glycophorin B and inhibits merozoite invasion in human red blood cells, Mol. Biochem. Parasitol., 183, 23-31. PMID: 22273481; PMCID: PMC3307866.
Brown, J.W., Farelli, J.D. & McKnight, C.J. (2011) On unsatisfied hydrogen bonds in the N-terminal subdomain of villin headpiece, J. Mol. Biol., 413, 543-547. PMID: 21903098; PMCID: PMC3196028.
Packer, L.E., Song, B., Raleigh, D.P. & McKnight, C.J. (2011) Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding, Biochem., 50, 3706-3712. PMID: 21449557; PMCID: PMC3086997.
Shukla, A.N., Daly, M.K., McKnight, C.J. & Freddo, T. (2011) Intraocular ophthalmic ointment following anterior segment surgery, J. Cataract Refract. Surg., 37, 2218-2221. PMID: 22001107.
Brown, J.W. & McKnight, C.J. (2010) Molecular model of the microvillar cytoskeleton and organization of the brush border, PLoS ONE, 5, e9406. PMID: 20195380; PMCID: PMC2827561.
Brown, J.W. & McKnight, C.J. (2010) Identifying competitive protein antagonists for F-actin with reverse-phase high-performance liquid chromatography, Anal. Biochem., 398, 117-119. PMID: 19932072; PMCID: PMC2812587.
Wang, L.B., Jiang, Z.G., McKnight, C.J. & Small, D.M. (2010) Interfacial Properties of Apolipoprotein B292-593 (B6.4-13) and B611-782 (B13-17). Insights into the Structure of the Lipovitellin Homology Region in Apolipoprotein B, Biochem., 49, 3898-3907. PMID: ISI:000277212400013.
Zhong, S.M., Magnolo, A.L., Sundaram, M., Zhou, H., Yao, E.F., Di Leo, E., Loria, P., Wang, S.A., Bamji-Mirza, M., Wang, L.S., McKnight, C.J., Figeys, D., Wang, Y.W., Tarugi, P. & Yao, Z.M. (2010) Nonsynonymous Mutations within APOB in Human Familial Hypobetalipoproteinemia EVIDENCE FOR FEEDBACK INHIBITION OF LIPOGENESIS AND POSTENDOPLASMIC RETICULUM DEGRADATION OF APOLIPOPROTEIN B, J. Biol. Chem., 285, 6453-6464. PMID: ISI:000275367500050.
Brown, J.W., 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. PMID: ISI:000271167400005.
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 Sci., 18, 629-636. PMID: 19241372; PMCID: PMC2760368.
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, Clin. Chim. Acta, 403, 254-256. PMID: 19306858.
Meng, J. & McKnight, C.J. (2009) Heterogeneity and dynamics in villin headpiece crystal structures, Acta Crystallogr. D Biol. Crystallogr., 65, 470-476. PMID: 19390152; PMCID: PMC2672817.
Mitsche, M.A., 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), Langmuir, 25, 2322-2330. PMID: 19146422.
Vugmeyster, L. & McKnight, C.J. (2009) Phosphorylation-induced changes in backbone dynamics of the dematin headpiece C-terminal domain, J. Biomol. NMR, 43, 39-50. PMID: 19030997; PMCID: PMC2796552.
Jiang, Z.G., Liu, Y., Hussain, M.M., Atkinson, D. & McKnight, C.J. (2008) Reconstituting initial events during the assembly of apolipoprotein B-containing lipoproteins in a cell-free system, J. Mol. Biol., 383, 1181-1194. PMID: 18804479; PMCID: PMC2637522.
Meng, J. & McKnight, C.J. (2008) Crystal structure of a pH-stabilized mutant of villin headpiece, Biochem., 47, 4644-4650. PMID: 18370407.
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. PMID: 18057002.
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, Biophys. J., 95, 5941-5950. PMID: 18820237; PMCID: PMC2599813.
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