Human CSTF2 RNA Recognition Motif Domain Binds to a U-Rich RNA Sequence through a Multistep Binding Process.

The RNA recognition motif (RRM) is a conserved and ubiquitous RNA-binding domain that plays essential roles in mRNA splicing, polyadenylation, transport, and stability. RRM domains exhibit remarkable diversity in binding partners, interacting with various sequences of single- and double-stranded RNA, despite their small size and compact fold. During pre-mRNA cleavage and polyadenylation, the RRM domain from CSTF2 recognizes U- or G/U-rich RNA sequences downstream from the cleavage and polyadenylation site to regulate the process.

Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.

NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling.

Structural features of Dnase1L3 responsible for serum antigen clearance.

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.

Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation.

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 (CSTF2) and showed that it changed the on-rate of RNA binding, leading to alternative polyadenylation in brains of mice carrying the same mutation.

LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex.

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.

Biochemical and structural characterization of analogs of MRE11 breast cancer-associated mutant F237C.

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.

The dynamic nature of the Mre11-Rad50 DNA break repair complex.

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.

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.

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.

A Survey of Reported Disease-Related Mutations in the MRE11-RAD50-NBS1 Complex.

The MRE11-RAD50-NBS1 (MRN) protein complex is one of the primary vehicles for repairing DNA double strand breaks and maintaining the genomic stability within the cell. The role of the MRN complex to recognize and process DNA double-strand breaks as well as signal other damage response factors is critical for maintaining proper cellular function. Mutations in any one of the components of the MRN complex that effect function or expression of the repair machinery could be detrimental to the cell and may initiate and/or propagate disease.

Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions.

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.

Adjacent mutations in the archaeal Rad50 ABC ATPase D-loop disrupt allosteric regulation of ATP hydrolysis through different mechanisms.

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.

A DNA aptamer reveals an allosteric site for inhibition in metallo-β-lactamases.

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.

Increasing the buffering capacity of minimal media leads to higher protein yield.

We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in Escherichia coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins, we found a clear correlation between the protein yield and change in media pH over time, where the minimal media that were the most buffered and therefore most resistant to change in pH produced the most protein.

Methyl-Based NMR Spectroscopy Methods for Uncovering Structural Dynamics in Large Proteins and Protein Complexes.

NMR spectroscopy is particularly adept at site-specifically monitoring dynamic processes in proteins, such as protein folding, domain movements, ligand binding, and side-chain rotations. By coupling the favorable spectroscopic properties of highly dynamic side-chain methyl groups with transverse-relaxation-optimized spectroscopy (TROSY), it is now possible to routinely study such dynamic processes in high-molecular-weight proteins and complexes approaching 1 MDa. In this Perspective, we describe many elegant methyl-based NMR experiments that probe slow (second) to fast (picosecond) dynamics in large systems.

The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity.

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 RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF-77 and CstF-50.

A dynamic allosteric pathway underlies Rad50 ABC ATPase function in DNA repair.

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.

ClpB N-terminal domain plays a regulatory role in protein disaggregation.

ClpB/Hsp100 is an ATP-dependent disaggregase that solubilizes and reactivates protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. The ClpB-substrate interaction is mediated by conserved tyrosine residues located in flexible loops in nucleotide-binding domain-1 that extend into the ClpB central pore. In addition to the tyrosines, the ClpB N-terminal domain (NTD) was suggested to provide a second substrate-binding site; however, the manner in which the NTD recognizes and binds substrate proteins has remained elusive.

Structure-Based Assignment of Ile, Leu, and Val Methyl Groups in the Active and Inactive Forms of the Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 2.

Resonance assignments are the first step in most NMR studies of protein structure, function, and dynamics. Standard protein assignment methods employ through-bond backbone experiments on uniformly (13)C/(15)N-labeled proteins. For larger proteins, this through-bond assignment procedure often breaks down due to rapid relaxation and spectral overlap.

A similar in vitro and in cell lysate folding intermediate for the FF domain.

Understanding the mechanisms by which proteins fold into their three-dimensional structures, including a description of the intermediates that are formed during the folding process, remains a goal of protein science. Most studies are performed under carefully controlled conditions in which the folding reaction is monitored in a buffer solution that is far from the natural milieu of the cell. Here, we have used (13)C and (1)H relaxation dispersion NMR spectroscopy to study folding of the FF domain in both Escherichia coli and Saccharomyces cerevisiae cellular lysates.

Viscosity-dependent kinetics of protein conformational exchange: microviscosity effects and the need for a small viscogen.

Conformational rearrangements are critical to a variety of biological processes including protein folding and misfolding, ligand binding, enzyme catalysis, and signal transduction. Viscosity-dependent kinetics measurements can provide crucial insights into the dynamics of protein conformational exchange by highlighting the relative importance of frictional forces derived from either solvent or from internal protein interactions in activating the exchange reaction. Here, we analyze the kinetics of interconversion between the native and intermediate states of the four helix bundle FF domain recorded in solutions containing the viscogens glycerol or bovine serum albumin (BSA), using the viscosity measured from the translational diffusion of probes of different sizes.

Understanding the mechanism of proteasome 20S core particle gating.

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood.