Biology subjects

Fish, A.

Publications and source records attributed to Fish, A..

3 recordsLinked to original sources

bioRxiv2018

Lysophosphatidic acid produced by Autotaxin acts as an allosteric modulator of its catalytic efficiency

Autotaxin is a secreted phosphodiesterase that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA). LPA controls key cellular responses such as migration, proliferation and survival, implicating ATX-LPA signalling in various (patho)physiological processes and establishing it as a drug target. ATX structural and functional studies have revealed an orthosteric and an allosteric site, the \"pocket\" and the \"tunnel\". Here, we revisit the kinetics of the ATX catalytic cycle in light of allosteric regulation, dissecting the different steps and pathways that lead to LPC hydrolysis. Consolidating all experimental kinetics data to a comprehensive catalytic model supported by molecular modelling simulations, suggests a positive feedback mechanism, regulated by the abundance of the LPA products activating hydrolysis of different LPC species. Our results complement and extend current understanding of ATX hydrolysis in light of the allosteric regulation by produced LPA species, and have implications for the design and application of orthosteric and allosteric ATX inhibitors.

biochemistry↗

bioRxiv2018

Characterisation and structure determination of a llama-derived nanobody targeting the J-base binding protein 1

The J-base Binding Protein 1 (JBP1) contributes to biosynthesis and maintenance of base J ({beta}-D-glucosyl-hydroxymethyluracil), a modification of thymidine confined to some protozoa. Camelid (llama) single domain antibody fragments (nanobodies) targeting JBP1 were produced for use as crystallization chaperones. Surface plasmon resonance (SPR) screening identified Nb6 as a strong binder, recognising JBP1 with a 1:1 stoichiometry and high affinity (kD=30nM). Crystallisation trials of JBP1 in complex with Nb6, yielded crystals diffracting to 1.47[A] resolution.\n\nHowever, the asymmetric unit dimensions and molecular replacement with a nanobody structure, clearly showed that the crystals of the expected complex with JBP1 were of the nanobody alone. Nb6 crystallizes in spacegroup P31 with two molecules in the asymmetric unit; its crystal structure was refined to a final resolution of 1.64[A]. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.\n\nSynopsisA camelid single domain antibody fragment (nanobody) is shown to have high affinity towards its recognition target, the J-base binding protein 1 (JBP1). The serendipitous crystallisation of this nanobody alone, and its crystal structure solution and refinement to 1.64[A] resolution are described. Ensemble refinement suggests that on the ligand-free state one of the complementarity determining regions (CDRs) is flexible while the other two adopt well-defined conformations.

biochemistry↗

bioRxiv2018

Dynamic Kinetochore Size Regulation Promotes Microtubule Capture And Chromosome Biorientation In Mitosis

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. An outer layer of the kinetochore known as the fibrous corona transiently expands in early mitosis and disassembles upon microtubule capture. Neither the functional importance nor the mechanistic basis for this are known. Here we show that the dynein adaptor Spindly and the RZZ kinetochore complex drive fibrous corona formation in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ:Spindly complexes into a corona-like meshwork in cells and in vitro. Concurrent with corona expansion, Spindly potentiates corona shedding by recruiting dynein via three conserved short linear motifs. Expanded, non-sheddable fibrous coronas engage in extensive, long-lived lateral microtubule interactions that persist to metaphase and result in fused sister kinetochores, formation of merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

cell biology↗