Structure-guided reshaping of the acyl binding pocket of 'TesA thioesterase enhances octanoic acid production in E. coli.

Medium-chain fatty acids (C6-C10) have attracted much attention recently for their unique properties compared to their long-chain counterparts, including low melting points and relatively higher carbon conversion yield. Thioesterase enzymes, which can catalyze the hydrolysis of acyl-ACP (acyl carrier protein) to release free fatty acids (FAs), regulate both overall FA yields and acyl chain length distributions in bacterial and yeast fermentation cultures. These enzymes typically prefer longer chain substrates. Herein, seeking to increase bacterial production of MCFAs, we conducted structure-guided mutational screening of multiple residues in the substrate-binding pocket of the E. coli thioesterase enzyme 'TesA. Confirming our hypothesis that enhancing substrate selectivity for medium-chain acyl substrates would promote overall MCFA production, we found that replacement of residues lining the bottom of the pocket with more hydrophobic residues strongly promoted the C8 substrate selectivity of 'TesA. Specifically, two rounds of saturation mutagenesis led to the identification of the 'TesARD-2 variant that exhibited a 133-fold increase in selectivity for the C8-ACP substrate as compared to C16-ACP substrate. Moreover, the recombinant expression of this variant in an E. coli strain with a blocked ß-oxidation pathway led to a 1030% increase in the in vivo octanoic acid (C8) production titer. When this strain was fermented in a 5-L fed-batch bioreactor, it produced 2.7 g/L of free C8 (45%, molar fraction) and 7.9 g/L of total free FAs, which is the highest-to-date free C8 titer to date reported using the E. coli type II fatty acid synthetic pathway. Thus, reshaping the substrate binding pocket of a bacterial thioesterase enzyme by manipulating the hydrophobicity of multiple residues altered the substrate selectivity and therefore fatty acid product distributions in cells. Our study demonstrates the relevance of this strategy for increasing titers of industrially attractive MCFAs as fermentation products.

Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.

Structural analysis of fungal CENP-H/I/K homologs reveals a conserved assembly mechanism underlying proper chromosome alignment.

The kinetochore is a proteinaceous complex that is essential for proper chromosome segregation. As a core member of the inner kinetochore, defects of each subunit in the CENP-H/I/K complex cause dysfunction of kinetochore that leads to chromosome mis-segregation and cell death. However, how the CENP-H/I/K complex assembles and promotes kinetochore function are poorly understood. We here determined the crystal structures of CENP-I N-terminus alone from Chaetomium thermophilum and its complex with CENP-H/K from Thielavia terrestris, and verified the identified interactions. The structures and biochemical analyses show that CENP-H and CENP-K form a heterodimer through both N- and C-terminal interactions. CENP-I integrates into the CENP-H/K complex by binding to the C-terminus of CENP-H, leading to formation of the ternary complex in which CENP-H is sandwiched between CENP-K and CENP-I. Our sequence comparisons and mutational analyses showed that this architecture of the CENP-H/I/K complex is conserved in human. Mutating the binding interfaces of CENP-H for either CENP-K or CENP-I significantly reduced their localizations at centromeres and induced massive chromosome alignment defects during mitosis, suggesting that the identified interactions are critical for CENP-H/I/K complex assembly at the centromere and kinetochore function. Altogether, our findings unveil the evolutionarily conserved assembly mechanism of the CENP-H/I/K complex that is critical for proper chromosome alignment.