MDC1 (mediator of DNA damage checkpoint protein 1) regulates the recognition and repair of DNA double strand breaks in mammalian cells through its interactions with nuclear foci containing the COOH-terminally phosphorylated form of the histone variant, H2AX. Here we demonstrate that the tandem BRCT repeats of MDC1 directly bind to the phosphorylated tail of H2AX-Ser(P)-Gln-Glu-Tyr, in a manner that is critically dependent on the free carboxylate group of the COOH-terminal Tyr residue. We have determined the x-ray crystal structure of the MDC1 BRCT repeats at 1.45 Å resolution. By a comparison with the structure of the BRCA1 BRCT bound to a phosphopeptide, we suggest that two arginine residues in MDC1, Arg 1932 and Arg 1933 may recognize the COOH terminus of the peptide as well as the penultimate Glu of H2AX, while Gln 2013 may provide additional specificity for the COOH-terminal Tyr.Tandem BRCT 3 repeats, initially discovered at the COOH terminus of the breast cancer-associated protein 1, BRCA1, are phosphoprotein recognition modules that play key signaling roles in the cellular response to DNA damage (1). Individual repeats are ϳ90 -100 amino acids in size. While they can fold independently, they often exist in tandem pairs where they pack in a head-tail manner (2-4). The BRCT repeats of BRCA1 have been shown to specifically bind to pSer-X-X-Phe peptide targets, such as the BACH1 helicase (5, 6), and the transcriptional co-repressor CtIP (7). Structural studies reveal that the NH 2 -terminal BRCT repeat is responsible for phosphoserine recognition, while the phenylalanine side chain is recognized by a pocket at the interface between the NH 2 -and COOH-terminal repeats (8 -10). Mutations that perturb the phenylalanine recognition pocket disrupt phosphopeptide binding and explain the enhanced cancer risks associated with some of these mutations.MDC1 (mediator of DNA damage checkpoint protein 1) is another BRCT repeat protein that plays a critical role in the DNA damage response. MDC1 has been implicated in the recognition and repair of DNA double strand breaks through its rapid co-localization with ␥-H2AX, the COOH-terminally phosphorylated form of the histone variant H2AX, at the sites of double strand breaks in mammalian nuclei (11,12). MDC1 also facilitates the recruitment of other repair proteins to these foci, including the MRE11 complex and the BRCT proteins 53BP1 and BRCA1, and is required for the efficient repair of ionizing radiation-induced DNA damage. The COOH-terminal BRCT repeats of MDC1 can specifically bind to phosphopeptides with specificity for a tyrosine residue at the ϩ3 position relative to the phosphoserine and some specificity for a glutamic acid at the ϩ2, matching the sequence of the ␥-H2AX tail: Ser(P)-Gln-Glu-Tyr (13). Here we demonstrate that the MDC1 BRCT repeats bind to the ␥-H2AX tail in a manner that is critically dependent on the free carboxylate group of the COOH-terminal tyrosine residue. The crystal structure of the MDC1 BRCT repeats reveals a phosphoserine binding pocket and ad...

MicroRNAs (miRNAs) regulate gene expression in a variety of biological pathways such as development and tumourigenesis. miRNAs are initially expressed as long primary transcripts (pri-miRNAs) that undergo sequential processing by Drosha and then Dicer to yield mature miRNAs. miR-17~92 is a miRNA cluster that encodes 6 miRNAs and while it is essential for development it also has reported oncogenic activity. To date, the role of RNA structure in miRNA biogenesis has only been considered in terms of the secondary structural elements required for processing of pri-miRNAs by Drosha. Here we report that the miR-17~92 cluster has a compact globular tertiary structure where miRNAs internalized within the core of the folded structure are processed less efficiently than miRNAs on the surface of the structure. Increased miR-92 expression resulting from disruption of the compact miR-17~92 structure results in increased repression of integrin α5 mRNA, a known target of miR-92a. In summary, we describe the first example of pri-miRNA structure modulating differential expression of constituent miRNAs.

CpxP is a novel bacterial periplasmic protein with no homologues of known function. In Gram-negative enteric bacteria, CpxP is thought to interact with the two-component sensor kinase, CpxA, to inhibit induction of the Cpx envelope stress response in the absence of protein misfolding. CpxP has also been shown to facilitate DegP-mediated proteolysis of misfolded proteins. Six mutations that negate the ability of CpxP to function as a signaling protein are localized in or near two conserved LTXXQ motifs that define a class of proteins with similarity to CpxP, Pfam PF07813. To gain insight into how these mutations might affect CpxP signaling and/or proteolytic adaptor functions, the crystal structure of CpxP from Escherichia coli was determined to 2.85-Å resolution. The structure revealed an antiparallel dimer of intertwined ␣-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold, with the conserved LTXXQ motifs forming two diverging turns at one end. Biochemical studies demonstrated that CpxP maintains a dimeric state but may undergo a slight structural adjustment in response to the inducing cue, alkaline pH. Three of the six previously characterized cpxP loss-of-function mutations, M59T, Q55P, and Q128H, likely result from a destabilization of the protein fold, whereas the R60Q, D61E, and D61V mutations may alter intermolecular interactions.

Retromer is a peripheral membrane protein complex that plays a critical role in a broad range of physiological, developmental and pathological processes by mediating retrograde transport of proteins from endosomes to the trans-Golgi (TGN) network. Wnt signalling, toxin transport and amyloid production in Alzheimer's disease are just some of the processes known to be regulated by retromer-mediated trafficking. Mammalian retromer consists of a core heterotrimeric cargorecognition subcomplex associated with a membrane-targeting dimer of sorting nexins. The core subcomplex consists of vacuolar protein sorting (VPS)26, VPS29 and VPS35 subunits that play different roles in complex stabilisation and in binding to transport cargo and regulatory proteins. The composition of the membrane-binding subcomplex is inconclusive but consists of a homo-or heterodimer of sorting nexins, SNX1, SNX2, SNX5 and SNX6. These BAR (Bin/Amphiphysin/Rvs)domain proteins can induce the formation of high curvature membrane tubules through the formation of a polymerised helical coat. We have used small-angle X-ray scattering (SAXS), X-ray crystallography, nuclear magnetic resonance (NMR) and isothermal titration calorimetry to elucidate a qualitative and quantitative model of retromer assembly [1]. In our proposed model, VPS35 forms an extended, gently curved structure composed of alternating HEAT-like helical repeats. VPS26 and VPS29 bind to distal ends through N-and C-terminal regions of VPS35, respectively, to form a stable trimeric core assembly. Results from thermodynamics experiments have shown that VPS29 and VPS26 bind to VPS35 completely independently of each other, confirming that VPS35 plays the role of central scaffold and VPS29 and VPS26 do not form any contact with each other. Intriguingly, the core trimeric complex is able to form a symmetric dimer, which may have implications for functional interactions in vivo. Solution structures of SNX1 and SNX2 homodimers have been determined using SAXS. The SNX dimer associates with cellular membranes enriched in phosphatidylinositol 3-phosphate and 3,5diphosphate and is believed to polymerise into a helical protein coat. NMR studies have confirmed that VPS29 coordinates the binding of the core subcomplex to this membrane remodelling complex. This coupling of cargo binding and membrane tubulating events enable retromer to recruit cargo molecules into a tubular endosome-to-Golgi transport carrier.

No abstract