Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD(+) is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD(+) ratio. Here we investigate the molecular mechanism for NADH/NAD(+) sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD(+), (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40 degrees closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.

Purine interruptions of polypyrimidine (Py) tract splice site signals contribute to human genetic diseases. The essential splicing factor U2AF 65 normally recognizes a Py tract consensus sequence preceding the major class of 3′ splice sites. We found that neurofibromatosisor retinitis pigmentosa-causing mutations in the 5′ regions of Py tracts severely reduce U2AF 65 affinity. Conversely, we identified a preferred binding site of U2AF 65 for purine substitutions in the 3′ regions of Py tracts. Based on a comparison of new U2AF 65 structures bound to either A-or G-containing Py tracts with previously identified pyrimidine-containing structures, we expected to find that a D231V amino acid change in U2AF 65 would specify U over other nucleotides. We found that the crystal structure of the U2AF 65 -D231V variant confirms favorable packing between the engineered valine and a target uracil base. The D231V amino acid change restores U2AF 65 affinity for two mutated splice sites that cause human genetic diseases and successfully promotes splicing of a defective retinitis pigmentosa-causing transcript. We conclude that reduced U2AF 65 binding is a molecular consequence of disease-relevant mutations, and that a structure-guided U2AF 65 variant is capable of manipulating gene expression in eukaryotic cells.pre-mRNA splicing | protein-RNA complex | protein engineering | crystal structure | RRM A pproximately 15% of the documented disease-causing point mutations disrupt consensus splice site elements in premRNAs, including a polypyrimidine (Py) tract between a branch point sequence (BPS) and an AG dinucleotide at the junction of the 3′ splice site (1) (Fig. 1A). For example, disease-causing mutations in Py tracts have been documented in ∼3,000 genes in the Human Gene Mutation Database (2), and an estimated 20% of these mutations affect regulatory splice site signals (3, 4). One of the earliest reports of a splice site mutation as a major cause of inherited human disease was for β-thalassemia (reviewed in ref. 5), for which splice site mutations in the human β-globin gene (HBB) are found in ∼14% of patients, causing symptoms of mild to severe anemia (reviewed in ref. 6).With the emergence of high-throughput sequencing technologies, splice site mutations in specific transcripts have been identified as common contributors to neuromuscular disorders, metabolic disorders, cancers, leukemias, deafness, and blindness, among other disorders (reviewed in ref. 4). Retinitis pigmentosa, the most prevalent form of inherited blindness in adults, represents one such disease that is primarily the consequence of mutations in splice sites of vision-relevant transcripts or splicing factors responsible for their recognition (reviewed in ref. 7). Neurofibromatosis type I, a disease characterized by tumors of nerve tissue, is an inherited disorder in which nearly 30% of the documented mutations disrupt neurofibromin 1 (NF1) splice sites (reviewed in ref. 8). Despite etiologic progress, the relationships between disease-causing pre-mRNA splice s...

The RNA recognition motif (RRM) is a prevalent class of RNA binding domain. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively few. Here, we use isothermal titration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF 65 , (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (−30 to −60 kcal mol −1 ) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearly physiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs.The RNA recognition motif (RRM) is an abundant domain among proteins with central roles in post-transcriptional gene regulation (1) (Figure 1). Multiple RRMs often occur per polypeptide. For example, tandem RRMs of the splicing factors U2AF 65 , TIA-1 and SXL recognize U-rich pre-mRNA sites (2,3). The poly(A) binding protein (PAB) enhances translation following RRM-mediated recognition of the mRNA tail (4). Beyond the wellestablished role of canonical RRMs to bind RNA, RRM variants such as the U2AF Homology Motif (UHM) are dedicated to protein-protein interactions (5). This breadth of functions illustrates the importance of elucidating the structural and thermodynamic forces responsible for RRM interactions.Despite extensive structural and functional investigations of RRMs, full thermodynamic characterizations of the enthalpy and entropy changes during RRM/RNA binding are scarce. A thorough characterization of RNA binding by an RRM has been completed for the U1A splicing factor (6). However, this rare example focuses on a single RRM binding a singlestranded (ss)RNA site within a stem loop, which differs from the prevalent systems of multiple RRMs recognizing unstructured ssRNAs. Enthalpy and entropy changes of ssRNA binding have been determined for a number of unrelated domains, including Hfq, GLD-1, trp attenuation protein (TRAP), tristetraprolin, and T4 translational regulatory protein (7)(8)(9)(10)(11). Given the diversity of these domain classes, it is unsurprising that no common thermodynamic themes of ssRNA binding have emerged to date. In contrast, protein/protein, protein/ligand, and protein/DNA interactions have been investigated in depth.Previously, we found a large favorable enthalpy change (ΔH) and a large unfavorable entropy change (−TΔS) for poly(U) recognition by the tandem RRMs of U2AF 65 (ΔH −70 † This work was supported by the National Institutes of Health (Grant GM070503 to C.L.K.). Figure 1C). To enable future comparison with higher order complexes, the U2AF 65 construct includes a Cterminal UHM that lacks detectable RNA affinity (5).The ability of U2AF 65 , SXL, and TIA-1 to recognize comparable U-rich sequences offered the opportunity to compare various RRM-containing proteins binding identical RNA sites (3,15). We preferred a poly(U) binding site for ITC, sinc...

Undergraduates in biology and chemistry encounter images of protein structures, solved by X-ray crystallography, but are often ill equipped to interpret and use these images in their education. A simple computer-based lab activity is presented and described here that introduces students to the origin of X-ray crystallographic images and allows them to experience the basics of protein model building, through the use of lysozyme. The lab activity also directs students to examine lysozyme’s role as a crystallization aid in the solution of a G-protein-coupled receptor. Students learn to use model building and visualization programs, Coot and PyMOL, along with the RCSB Protein Data Bank, to obtain, use and interpret electron density maps with structure files to assess a protein model.

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