NMR studies of Dynamics in Large Protein Complexes
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.
DNA Damage Repair Response
The body experiences millions of lesions to its genomic DNA everyday from variety of internal and external factors and so our cells have evolved complex systems to monitor and preserve the integrity of our DNA. Incorrectly repairing or failure to repair a DNA break can result in mutations and genomic instability, potentially leading to carcinogenesis. In fact, failure of even one DNA repair pathway can lead to various types of cancers. Detecting and coordinating the repair of DNA double strand breaks (DSBs) starts with the essential Mre11-Rad50-Nbs1 (MRN) protein complex. MRN uses ATP-dependent conformational changes to control its various repair activities: tethering the broken DNA ends, unwinding the DNA duplex, processing the DNA ends to prepare for repair, and recruiting downstream signaling effectors to the site of damage.
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Mre11-Rad50-DNA complex
We are using methyl-based NMR techniques to study the core Mre11-Rad50 (MR) complex bound to DNA DSB substrates. The ATPase activity of Rad50 regulates the conformation of MR: binding of ATP results in a closed MR structure, which opens to a more extended structure upon ATP hydrolysis. While both conformations can bind to DNA, only the open state is capable of Mre11 nuclease activity. In fact, our structural studies suggest that a heterogeneous ensemble of MR-DNA conformations coordinates its various functions. With NMR as a tool, we can site-specifically monitor protein motions over the picosecond to days timescale and we are able to 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: RNA cleavage and polyadenylation
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 presented only in the male members of a family in Morocco, with failure to attain developmental milestones, low IQs, and evidence of autism symptoms. Using NMR, we solved the structure of the D50A RRM and are studying the RNA binding affinity and the structure-function relationship in wild type and mutated CstF-64 RRMs, and how mutant dynamics change upon binding to GU-rich sequence elements.
CRES functional amyloid
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 alongside the potential of CRES binding to and templating off extracellular DNA, thereby altering the kinetics of assembly.
