Molecular Basis of eRF3 Recognition by the MLLE Domain of Poly(A)-Binding Protein

PABPC1 (cytosolic poly(A)-binding protein 1) is an RNA-binding protein that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. In addition to RNA-binding domains, PABPC1 contains a unique protein-protein interaction domain, MLLE (also known as PABC) that binds regulatory proteins and translation factors that contain a conserved 12 amino acid peptide motif termed PAM2. Eukaryotic Release Factor 3 (eRF3/GSPT1) contains two overlapping PAM2 sequences, which are required for its activity. Here, we determined the crystal structures of the MLLE domain from PABPC1 in complex with the two PAM2 regions of eRF3. The structures reveal a mechanism of cooperativity between the two PAM2 sites that increases the binding affinity but prevents the binding of more than one molecule of eRF3 to PABPC1. Relative to previous structures, the high-resolution crystal structures force a re-evaluation of the PAM2 motif and improve our understanding of the molecular basis of MLLE peptide recognition

Structure of GlgS from Escherichia coli suggests a role in protein–protein interactions

BACKGROUND: The Escherichia coli protein GlgS is up-regulated in response to starvation stress and its overexpression was shown to stimulate glycogen synthesis. RESULTS: We solved the structure of GlgS from E. coli, a member of an enterobacterial protein family. The protein structure represents a bundle of three α-helices with a short hydrophobic helix sandwiched between two long amphipathic helices. CONCLUSION: GlgS shows structural homology to Huntingtin, elongation factor 3, protein phosphatase 2A, TOR1 motif domains and tetratricopeptide repeats, suggesting a possible role in protein–protein interactions

Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity

Connecdenn, a novel DENN domain-containing protein of neuronal clathrin-coated vesicles functioning in synaptic vesicle endocytosis

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the α-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three α-ear-binding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis

Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase

With the increased incidence of tuberculosis (TB) caused by Mycobacterium tuberculosis there is an urgent need for new and better anti-tubercular drugs. N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a key enzyme in the succinylase pathway for the biosynthesis of meso-diaminopimelic acid (meso-DAP) and L-lysine. DapE is a zinc containing metallohydrolase which hydrolyses N-succinyl L,L diaminopimelic acid (L,L-NSDAP) to L,L-diaminopimelic acid (L,L-DAP) and succinate. M. tuberculosis DapE (MtDapE) was cloned, over-expressed and purified as an N-terminal hexahistidine ((His)6) tagged fusion containing one zinc ion per DapE monomer. We redesigned the DAP synthetic pathway to generate L,L-NSDAP and other L,L-NSDAP derivatives and have characterised MtDapE with these substrates. In contrast to its other Gram negative homologues, the MtDapE was insensitive to inhibition by L-captopril which we show is consistent with novel mycobacterial alterations in the binding site of this drug

H(C)CH-COSY and (H)CCH-COSY experiments for 13C-labeled proteins in H2O solution

UI - 99018160NRC publication: Ye

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

NRC publication: Ye