Vasohibins are tubulin tyrosine carboxypeptidases that are important for neuron physiology. We solved crystal structures of human vasohibin 1 and 2 in complex with small vasohibin-binding protein (SVBP) in the absence and presence of different inhibitors and a C-terminal -tubulin peptide. In combination with functional data, we propose that SVBP acts as an activator of vasohibins. An extended groove and a distinctive surface residue patch of vasohibins define the specificity determinants for recognizing and cleaving the C-terminal tyrosine of -tubulin and for binding microtubules, respectively. The vasohibin-SVBP interaction and the ability of the enzyme complex to associate with microtubules regulate axon specification of neurons. Our results define the structural basis of tubulin detyrosination by vasohibins and show the relevance of this process for neuronal development. They further offer a unique platform for developing drugs against human conditions with abnormal tubulin tyrosination levels including cancer, heart defects and possibly brain disorders.

Summary From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.

The YTH domain-containing protein Mmi1, together with other factors, constitutes the machinery used to selectively remove meiosis-specific mRNA during the vegetative growth of fission yeast. Mmi1 directs meiotic mRNAs to the nuclear exosome for degradation by recognizing their DSR (determinant of selective removal) motif. Here, we present the crystal structure of the Mmi1 YTH domain in the apo state and in complex with a DSR motif, demonstrating that the Mmi1 YTH domain selectively recognizes the DSR motif. Intriguingly, Mmi1 also contains a potential m6A (N6-methyladenine)-binding pocket, but its binding of the DSR motif is dependent on a long groove opposite the m6A pocket. The DSR-binding mode is distinct from the m6A RNA-binding mode utilized by other YTH domains. Furthermore, the m6A pocket cannot bind m6A RNA. Our structural and biochemical experiments uncover the mechanism of the YTH domain in binding the DSR motif and help to elucidate the function of Mmi1.

The abnormal expression of Annexin II (AnxA2, A2) has been associated with the development of tumors; however, its expression and function in multiple myeloma (MM) is less known. We compared the expression of AnxA2 in primary myeloma cells from MM patients with that in normal plasma cells from normal subjects and found that myeloma cells from patients had higher expression of AnxA2. Expression of AnxA2 was also significantly higher in MM cell lines U266 and RPMI8226, compared with other hematologic tumor cell lines. Transfecting U266 and RPMI8226 cells with the small interfering RNA (siRNA) that targets human AnxA2 led to significant downregulation of AnxA2 expression, which resulted in the decreased proliferation, invasive potential and increased apoptosis of U266 and RPMI8226 cell lines. Silencing AnxA2 gene by siRNA also inhibited the expression of pro-angiogenic molecules including VEGF-C, VEGF-R2, MMP-2, MMP-9, MT1-MMP and TIMP-2 in the two cell lines. Our data suggested that the AnxA2 is overexpressed in MM patients and myeloma cell lines U266 and RPMI8226, and that AnxA2 overexpression appeared to affect the proliferation, apoptosis, invasive potential and production of pro-angiogenic factors in MM cell lines U266 and RPMI8226.

HMCES and yedK were recently identified as sensors of abasic sites in ssDNA. In this study, we present multiple crystal structures captured in the apo-, nonspecific-substrate-binding, specific-substrate-binding, and product-binding states of yedK. In combination with biochemical data, we unveil the molecular basis of AP site sensing in ssDNA by yedK. Our results indicate that yedK has a strong preference for AP site-containing ssDNA over native ssDNA and that the conserved Glu105 residue is important for identifying AP sites in ssDNA. Moreover, our results reveal that a thiazolidine linkage is formed between yedK and AP sites in ssDNA, with the residues that stabilize the thiazolidine linkage important for the formation of DNA-protein crosslinks between yedK and the AP sites. We propose that our findings offer a unique platform to develop yedK and other SRAP domain-containing proteins as tools for detecting abasic sites in vitro and in vivo.