Latham Lab Projects

The characterization of large macromolecular assemblies can be challenging for the traditional structural biology techniques of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. Protein complexes are often connected via flexible linkers or intrinsically disordered domains and can undergo large-scale domain motions, which is sub-optimal for X-ray crystallography. Meanwhile the large sizes of these macromolecular assemblies makes traditional NMR techniques problematic. Novel solution state NMR methodologies have been developed that exploit the favorable spectroscopic properties of protein side chain methyl groups and have been successfully utilized to study the structure, function, and dynamics of protein complexes approaching 1 mega-Dalton. 

We have used methyl-based NMR techniques to study the core Mre11-Rad50 (MR) DNA damage repair protein complex bound to DNA double-stranded break substrates. The ATPase activity of Rad50 regulates the conformation of MR: binding of ATP results in a 'closed' MR structure, which opens to at least two 'open' structures upon ATP hydrolysis. While all of the conformations can bind to DNA, only the open state is capable of Mre11 nuclease activity. With NMR as a tool, we can site-specifically monitor protein motions over the picosecond to days timescale and study the protein dynamics within Mre11 (DNA unwinding), Rad50 (ATP hydrolysis), and the MR complex. We complement our NMR studies by using fluorescence, FRET, and yeast DNA repair assays to put our results into the full picture of the initial steps of the DNA damage repair response.

CstF-64 (encoding a 64 kDa protein) is a member of the Cleavage stimulation Factor (CstF) protein complex, which plays critical roles in transcription termination and mRNA 3’-end processing at polyadenylation sites. CstF-64 contains an RNA recognition motif (RRM) that binds to the G/U rich RNA sequences located downstream of the cleavage and polyadenylation site and hence is an important part in the regulation of these mRNA maturation processes. A single mutation in CstF-64, which converts an aspartate at amino acid position 50 to an alanine (D50A) in the RRM, has been discovered that caused severe intellectual disabilities in only the male members of a family in Morocco. Using NMR, we solved the structure of the D50A RRM and studied the RNA binding affinity and the structure-function relationship in wild type and mutated CstF-64 RRMs. We learned how mutant dynamics change upon binding to GU-rich sequence elements.

The epididymal lumen of the male reproductive tract contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation, sperm protection, and host-defense. This amyloid matrix contains several family 2 cystatins of cysteine protease inhibitors, including cystatin C and four members of the CRES (cystatin-related epididymal spermatogenic) sub-group. Using NMR, we solved the structure of CRES wild-type and found that CRES amyloid formation is complex and likely utilizes two mechanisms, including a unique interaction driven by changes in the structure of a CRES loop from a flexible linker in the monomer to a β-strand conformation in the advanced amyloid, as well as traditional domain swapping typical of other cystatins. We are studying the structural and dynamic characteristics of these two pathways as well as the role of extracellular DNA in CRES templating and kinetics of assembly.