Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.
Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full‐length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in‐solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration‐dependent manner, and its oligomerization is mediated via the positively charged N‐terminal microtubule‐binding domain. Our findings provide the first insights into the structure of full‐length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.
Polyglutamylation is a reversible post-translational modification that is catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Disheveled 3 (DVL3), thereby changing the interactome of DVL3, as well as increases its capacity to get phosphorylated, to undergo liquid-liquid phase separation (LLPS) and to act in the non-canonical Wnt pathway. Both carboxyterminal polyglutamylation and the resulting reduction in LLPS capacity of DVL3 were reverted by the deglutamylating enzyme CCP6, which demonstrates the causal relationship between TTLL11-mediated polyglutamylation and LLPS. We thus discovered a novel type of posttranslational modification, which significantly broadens the range of proteins that can be modified by polyglutamylation and provide first evidence that polyglutamylation can act as a regulator of protein LLPS.
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