Src homology 3 (SH3) domains are involved in the regulation of important cellular pathways, such as cell proliferation, migration and cytoskeletal modifications. Recognition of polyproline and a number of noncanonical sequences by SH3 domains has been extensively studied by crystallography, nuclear magnetic resonance and other methods. High-affinity peptides that bind SH3 domains are used in drug development as candidates for anticancer treatment. This review summarizes the latest achievements in deciphering structural determinants of SH3 function.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ ManNAc kinase (GNE/MNK), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (sialic acid). GNE/MNK is feedback inhibited by binding of the downstream product, CMP-sialic acid in its allosteric site. GNE mutations can result in two human disorders, hereditary inclusion body myopathy (HIBM) or sialuria. So far, no active site geometry predictions or conformational transitions involved with function are available for mammalian GNE/MNK. The N-terminal GNE domain is homologous to various prokaryotic 2-epimerases, some of which have solved crystallographic structures. The C-terminal MNK domain belongs to the sugar kinases superfamily; its crystallographic structure is solved at 2.84 A and three-dimensional structures have also been reported for several other kinases. In this work, we employed available structural data of GNE/MNK homologs to model the active sites of human GNE/MNK and identify critical amino acid residues responsible for interactions with substrates. In addition, we modeled effects of GNE/MNK missense mutations associated with HIBM or sialuria on helix arrangement, substrate binding, and enzyme action. We found that all reported mutations are associated with the active sites or secondary structure interfaces of GNE/MNK. The Persian-Jewish HIBM founder mutation p.M712T is located at the interface alpha4alpha10 and likely affects GlcNAc, Mg2+, and ATP binding. This work contributes to further understanding of GNE/MNK function and ligand binding, which may assist future studies for therapeutic options that target misfolded GNE/MNK in HIBM and/or sialuria.

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UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. In addition to the previously described 3 human GNE isoforms (hGNE1- hGNE3), our database and PCR analysis yielded an additional 5 human isoforms (hGNE4- hGNE8). hGNE1 is the ubiquitously expressed major isoform, while the hGNE2-8 isoforms are differentially expressed and may act as tissue-specific regulators of sialylation. hGNE2 and hGNE7 display a 31-residue N-terminal extension compared to hGNE1. Based on similarities to kinases and helicases, this extension does not seem to hinder the epimerase enzymatic active site. hGNE3 and hGNE8 contain a 55 residue N-terminal deletion, and a 50-residue N-terminal extension compared to hGNE1. The size and secondary structures of these fragments are similar, and modeling predicted that these modifications do not affect the overall fold compared to hGNE1. However, epimerase enzymatic activity of GNE3 and GNE8 is likely absent, since the deleted fragment contains important substrate binding residues in homologous bacterial epimerases. hGNE5-hGNE8 have a 53-residue deletion, which was assigned a role in substrate (UDP-GlcNAc) binding. Deletion of this fragment likely eliminates epimerase enzymatic activity. Our findings imply that GNE is subject to evolutionary mechanisms to increase cellular functions, without increasing the number of genes. Our expression and modeling data contribute to elucidation of the complex functional and regulatory mechanisms of human GNE, and may contribute to further elucidating the pathology and treatment strategies of the human GNE-opathies sialuria and hereditary inclusion body myopathy.

1Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and this interaction was blocked by TFP5, an inhibitor that prevents activation of Cdk5. In vitro peptide-based kinase assay revealed that four of six TRPA1 Cdk5 consensus sites acted as substrates for Cdk5, and modeling of the ankyrin repeats disclosed that phosphorylation would occur at characteristic pockets within the (T/S)PLH motifs. Calcium imaging of trigeminal ganglion neurons from genetically engineered mice overexpressing or lacking the Cdk5 activator p35 displayed increased or decreased responsiveness, respectively, to stimulation with the TRPA1 agonist allylisothiocyanate (AITC). AITCinduced chemo-nociceptive behavior was also heightened in vivo in mice overexpressing p35 while being reduced in p35 knockout mice. Our findings demonstrate that TRPA1 is a substrate of Cdk5 and that Cdk5 activity is also able to modulate TRPA1 agonist-induced calcium influx and chemo-nociceptive behavioral responses.Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that can modulate pain signaling 1,2 . Cdk5 is unlike other cyclin-dependent kinases because it is mostly active in post-mitotic neurons. Additionally, Cdk5 is not activated by a cyclin, but, instead, binds to two regulatory subunits, either p35 or p39, that are primarily restricted to neurons. Cdk5 activity is upregulated following peripheral administration of inflammatory agents such as carrageenan and Complete Freund's Adjuvant (CFA) 1,3 . Increased Cdk5 activity can, in turn, promote both mechanical and heat hyperalgesia 1,2 . Cdk5 activity can also affect morphine tolerance, where reduced Cdk5 activity correlates with delayed tolerance 4 . Therapeutically, intrathecal injection of the Cdk5 inhibitor roscovitine appears to attenuate CFA-induced heat hyperalgesia but not mechanical allodynia 3 while also reducing formalin-induced nociceptive behavior 5 . Cdk5 substrates include P/Q-type voltage-dependent calcium channels, which control calcium influx during nociception, and N-methyl-D-aspartate (NMDA) receptors, which relay pain signaling via the excitatory neurotransmitter glutamate 4 . Cdk5, however, is most directly linked to pain signaling by phosphorylating the pain transducing ion channel Transient Receptor Potential Vanilloid 1

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A straightforward method for predicting the protein structure is to find conformations that have the lowest energy along a chosen folding pathway. One approach in this direction is to produce a large number of structures by varying the dihedral angles of the molecule more or less randomly and then to screen each one using a suitable energy function. This procedure is computationally demanding, but by using a more realistic model, one hopes that the folding behavior one observes in calculations may better mimic the actual folding process in nature. The method is beginning to yield interesting results, thanks to the increase in the computational power but also to the intelligent selection of the folding pathway. This article reviews general features of this method, some important highlights of the particular procedure we used, and some of the more significant results obtained to date in our laboratory. The results are highly encouraging and indicate the direction of future effort that is most likely to be fruitful.

An approximate but rapid method for estimating hydrophobic energy is proposed. Aside from a scale factor, it is given by the pairwise sum of the surface area buried by each neighbor atom, but excluding those atoms in the same residue or in its sequence neighbor residues. This sum is found to be linearly related to the true buried area as calculated by the algorithm of Lee and Richards [1971, J. Mol. Biol., 55, 379-400], and to the contact potential of Miyazawa and Jernigan [1985, Macromolecules, 18, 534-552]. It correlates with experimental transfer free energies to approximately the same degree as that calculated using the true buried area. Furthermore, in a simple test of helix packing with ROP protein monomer, the new hydrophobic energy clearly discriminated one structure, with the lowest r.m.s. deviation from the crystal structure, against an exhaustive set of others.