Browsing by Author "Latham, Michael P. (TTU)"
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Item A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases(2019) Khan, Nazmul H. (TTU); Bui, Anthony A. (TTU); Xiao, Yang (TTU); Sutton, R. Bryan (TTU); Shaw, Robert W. (TTU); Wylie, Benjamin J. (TTU); Latham, Michael P. (TTU)The hydrolysis of β-lactam antibiotics by β-lactamase enzymes is the most prominent antibiotic resistance mechanism for many pathogenic bacteria. Out of this broad class of enzymes, metallo-β-lactamases are of special clinical interest because of their broad substrate specificities. Several in vitro inhibitors for various metallo-β-lactamases have been reported with no clinical efficacy. Previously, we described a 10-nucleotide single stranded DNA aptamer (10-mer) that inhibits Bacillus cereus 5/B/6 metallo-β-lactamase very effectively. Here, we find that the aptamer shows uncompetitive inhibition of Bacillus cereus 5/B/ 6 metallo-β-lactamase during cefuroxime hydrolysis. To understand the mechanism of inhibition, we report a 2.5 A resolution X-ray crystal structure and solution-state NMR analysis of the free enzyme. Chemical shift perturbations were observed in the HSQC spectra for several residues upon titrating with increasing concentrations of the 10-mer. In the X-ray crystal structure, these residues are distal to the active site, suggesting an allosteric mechanism for the aptamer inhibition of the enzyme. HADDOCK molecular docking simulations suggest that the 10-mer docks 26 A from the active site. We then mutated the three lysine residues in the basic binding patch to glutamine and measured the catalytic activity and inhibition by the 10-mer. No significant inhibition of these mutants was observed by the 10-mer as compared to wild type. Interestingly, mutation of Lys50 (Lys78; according to standard MBL numbering system) resulted in reduced enzymatic activity relative to wild type in the absence of inhibitor, further highlighting an allosteric mechanism for inhibition.Item A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair(2018) Boswell, Zachary K. (TTU); Rahman, Samiur (TTU); Canny, Marella D. (TTU); Latham, Michael P. (TTU)The Mre11-Rad50 protein complex is an initial responder to sites of DNA double strand breaks. Many studies have shown that ATP binding to Rad50 causes global changes to the Mre11-Rad50 structure, which are important for DNA repair functions. Here we used methyl-based NMR spectroscopy on a series of mutants to describe a dynamic allosteric pathway within Rad50. Mutations result in changes in the side chain methyl group chemical environment that are correlated with altered nanosecond timescale dynamics. We also observe striking relationships between the magnitude of chemical shift perturbations and Rad50 and Mre11 activities. Together, these data suggest an equilibrium between a ground state and an "active" dimerization competent state of Rad50 that has locally altered structure and dynamics and is poised for ATP-induced dimerization and eventual ATP hydrolysis. Thus, this sparsely populated intermediate is critical for Mre11-Rad50-directed DNA double strand break repair.Item A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3' end processing is associated with intellectual disability in humans(2020) Grozdanov, Petar N. (TTUHSC); Masoumzadeh, Elahe (TTU); Kalscheuer, Vera M.; Bienvenu, Thierry; Billuart, Pierre; Delrue, Marie Ange; Latham, Michael P. (TTU); MacDonald, Clinton C. (TTUHSC)CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the significance of 3' end mRNA processing in expression of genes important for brain plasticity and neuronal development.Item Adjacent mutations in the archaeal Rad50 ABC ATPase D-loop disrupt allosteric regulation of ATP hydrolysis through different mechanisms(2020) Boswell, Zachary K. (TTU); Canny, Marella D. (TTU); Buschmann, Tanner A. (TTU); Sang, Julie (TTU); Latham, Michael P. (TTU)DNA damage is the driving force for mutation and genomic instability, which can both lead to cell death or carcinogenesis. DNA double strand breaks are detected and processed in part by the Mre11-Rad50-Nbs1 protein complex. Although the Mre11-Rad50-Nbs1 complex is essential, several spontaneous mutations have been noted in various cancers. One of these mutations, within a conserved motif of Rad50, resulted in an outlier curative response in a clinical trial. We show through biochemical and biophysical characterization that this cancer-associated mutation and a second mutation to the adjacent residue, previously described in a breast cancer patient, both have gain-of-function Rad50 ATP hydrolysis activity that results not from faster association of the ATP-bound form but faster dissociation leading to less stable Rad50 dimer. This disruption impairs the regulatory functions of the protein complex leading to a loss of exonuclease activity from Mre11. Interestingly, these two mutations affect Rad50 structure and dynamics quite differently. These studies describe the relationship between function, structure, and molecular motions in improperly regulated Rad50, which reveal the underlying biophysical mechanism for how these two cancer-associated mutations affect the cell.Item Biochemical and structural characterization of analogs of MRE11 breast cancer-associated mutant F237C(2021) Rahman, Samiur (TTU); Beikzadeh, Mahtab (TTU); Latham, Michael P. (TTU)The MRE11–RAD50–NBS1 (MRN) protein complex plays a vital role in DNA double strand break sensing, signaling, and repair. Mutation in any component of this complex may lead to disease as disrupting DNA double strand break repair has the potential to cause translocations and loss of genomic information. Here, we have investigated an MRE11 mutation, F237C, identified in a breast cancer tumor. We found that the analogous mutant of Pyrococcus furiosus Mre11 diminishes both the exonuclease and endonuclease activities of Mre11 in vitro. Solution state NMR experiments show that this mutant causes structural changes in the DNA-bound Mre11 for both exo- and endonuclease substrates and causes the protein to become generally more rigid. Moreover, by comparing the NMR data for this cancer-associated mutant with two previously described Mre11 separation-of-nuclease function mutants, a potential allosteric network was detected within Mre11 that connects the active site to regions responsible for recognizing the DNA ends and for dimerization. Together, our data further highlight the dynamics required for Mre11 nuclease function and illuminate the presence of allostery within the enzyme.Item The dynamic nature of the Mre11-Rad50 DNA break repair complex(2021) Beikzadeh, Mahtab (TTU); Latham, Michael P. (TTU)The Mre11-Rad50-Nbs1/Xrs2 protein complex plays a pivotal role in the detection and repair of DNA double strand breaks. Through traditional and emerging structural biology techniques, various functional structural states of this complex have been visualized; however, relatively little is known about the transitions between these states. Indeed, it is these structural transitions that are important for Mre11-Rad50-mediated DNA unwinding at a break and the activation of downstream repair signaling events. Here, we present a brief overview of the current understanding of the structure of the core Mre11-Rad50 complex. We then highlight our recent studies emphasizing the contributions of solution state NMR spectroscopy and other biophysical techniques in providing insight into the structures and dynamics associated with Mre11-Rad50 functions.Item Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation(2022) Masoumzadeh, Elahe (TTU); Grozdanov, Petar N. (TTUHSC); Jetly, Anushka (TTU); MacDonald, Clinton C. (TTUHSC); Latham, Michael P. (TTU)Nascent pre-mRNA 3′-end cleavage and polyadenylation (C/P) involves numerous proteins that recognize multiple RNA elements. Human CSTF2 binds to a downstream U- or G/U-rich sequence through its RNA recognition motif (RRM) regulating C/P. We previously reported the only known disease-related CSTF2 RRM mutant (CSTF2D50A) and showed that it changed the on-rate of RNA binding, leading to alternative polyadenylation in brains of mice carrying the same mutation. In this study, we further investigated the role of electrostatic interactions in the thermodynamics and kinetics of RNA binding for the CSTF2 RRM and the downstream consequences for regulation of C/P. By combining mutagenesis with NMR spectroscopy and biophysical assays, we confirmed that electrostatic attraction is the dominant factor in RRM binding to a naturally occurring U-rich RNA sequence. Moreover, we demonstrate that RNA binding is accompanied by an enthalpy-entropy compensation mechanism that is supported by changes in pico-to-nanosecond timescale RRM protein dynamics. We suggest that the dynamic binding of the RRM to U-rich RNA supports the diversity of sequences it encounters in the nucleus. Lastly, in vivo C/P assays demonstrate a competition between fast, high affinity RNA binding and efficient, correct C/P. These results highlight the importance of the surface charge of the RRM in RNA binding and the balance between nascent mRNA binding and C/P in vivo.Item LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex(2022) Canny, Marella D. (TTU); Latham, Michael P. (TTU)The Mre11-Rad50-Nbs1 protein complex is one of the first responders to DNA double-strand breaks. Studies have shown that the catalytic activities of the evolutionarily conserved Mre11-Rad50 (MR) core complex depend on an ATP-dependent global conformational change that takes the macromolecule from an open, extended structure in the absence of ATP to a closed, globular structure when ATP is bound. We have previously identified an additional ‘partially open’ conformation using luminescence resonance energy transfer (LRET) experiments. Here, a combination of LRET and the molecular docking program HADDOCK was used to further investigate this partially open state and identify three conformations of MR in solution: closed, partially open, and open, which are in addition to the extended, apo conformation. Mutants disrupting specific Mre11-Rad50 interactions within each conformation were used in nuclease activity assays on a variety of DNA substrates to help put the three states into a functional perspective. LRET data collected on MR bound to DNA demonstrate that the three conformations also exist when nuclease substrates are bound. These models were further supported with small-angle X-ray scattering data, which corroborate the presence of multiple states in solution. Together, the data suggest a mechanism for the nuclease activity of the MR complex along the DNA.Item Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions(2020) Rahman, Samiur (TTU); Beikzadeh, Mahtab (TTU); Canny, Marella D. (TTU); Kaur, Navneet (TTU); Latham, Michael P. (TTU)Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11–Rad50–Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.Item Structural features of Dnase1L3 responsible for serum antigen clearance(2022) McCord, Jon J. (TTUHSC); Engavale, Minal (TTU); Masoumzadeh, Elahe (TTU); Villarreal, Johanna (TTUHSC); Mapp, Britney (TTU); Latham, Michael P. (TTU); Keyel, Peter A. (TTU); Sutton, R. Bryan (TTUHSC)Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 cannot rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are key structural differences between the two enzymes. First, Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. Second, the CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.Item The Functional Mammalian CRES (Cystatin-Related Epididymal Spermatogenic) Amyloid is Antiparallel β-Sheet Rich and Forms a Metastable Oligomer During Assembly(2019) Do, Hoa Quynh (TTUHSC); Hewetson, Aveline (TTUHSC); Myers, Caitlyn (TTUHSC); Khan, Nazmul H. (TTU); Hastert, Mary Catherine (TTU); M. Harsini, Faraz (TTUHSC); Latham, Michael P. (TTU); Wylie, Benjamin J. (TTU); Sutton, R. Bryan (TTUHSC); Cornwall, Gail A. (TTUHSC)An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of β-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel β-sheets instead of the more common parallel β-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel β-sheet-rich amyloids can be functional forms.Item The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity(2018) Grozdanov, Petar N. (TTUHSC); Masoumzadeh, Elahe (TTU); Latham, Michael P. (TTU); MacDonald, Clinton C. (TTUHSC)Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-rich sequences located downstream from the cleavage site through its RNAbinding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF- 77 and CstF-50. Here, we show that the carboxyterminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytailcarboxy- terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.