Structural basis for ESCRT-III CHMP3 recruitment of AMSH.

Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.

Structure and function of ESCRT-III.

ESCRT-III (endosomal sorting complex required for transport III) is required for the formation and abscission of intraluminal endosomal vesicles, which gives rise to multivesicular bodies, budding of some enveloped viruses and cytokinesis. ESCRT-III is composed of 11 members in humans, which, except for one, correspond to the six ESCRT-III-like proteins in yeast. At least CHMP (charged multivesicular body protein) 2A and CHMP3 assemble into helical tubular structures that provide a platform for membrane interaction and VPS (vacuolar protein sorting) 4-catalysed effects leading to disassembly of ESCRT-III CHMP2A-CHMP3 polymers in vitro. Progress towards the understanding of the structures and function of ESCRT-III, its activation, its regulation by accessory factors and its role in abscission of membrane enveloped structures in concert with VPS4 are discussed.

Structural basis for autoinhibition of ESCRT-III CHMP3.

Endosomal sorting complexes required for transport (ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III) are selectively recruited to cellular membranes to exert their function in diverse processes, such as multivesicular body biogenesis, enveloped virus budding, and cytokinesis. ESCRT-III is composed of members of the charged multivesicular body protein (CHMP) family--cytosolic proteins that are targeted to membranes via yet unknown signals. Membrane targeting is thought to result in a membrane-associated protein network that presumably acts at a late budding step. Here we provide structural evidence based on small-angle X-ray scattering data that ESCRT-III CHMP3 can adopt two conformations in solution: a closed globular form that most likely represents the cytosolic conformation and an open extended conformation that might represent the activated form of CHMP3. Both the closed and open conformations of CHMP3 interact with AMSH with high affinity. Although the C-terminal region of CHMP3 is required for AMSH interaction, a peptide thereof reveals only weak binding to AMSH, suggesting that other regions of CHMP3 contribute to the high-affinity interaction. Thus, AMSH, including its MIT (microtubule interacting and transport) domain, interacts with ESCRT-III CHMP3 differently from reported Vps4 MIT domain-CHMP protein interactions.

Ligation independent cloning (LIC) as a rapid route to families of recombinant biocatalysts from sequenced prokaryotic genomes.

A technique is presented for the high throughput generation of families of recombinant biocatalysts sourced from prokaryotic genomes, providing rapid access to the naturally evolved diversity of enzyme specificity for biocatalyst discovery. The method exploits a novel ligation independent cloning strategy, based on the locally engineered vector pET-YSBLIC and has been used for the rapid generation of a suite of expression plasmids containing genes encoding a family of six Baeyer-Villiger monooxygenases (BVMOs) from Mycobacterium tuberculosis H37Rv (MTb). The six resultant recombinant strains of E. coli B834 (DE3) expressing the genes were assayed for oxygenating activity in respect of the target reaction; the resolution of bicyclo[3.2.0]hept-2-en-6-one. The analysis of biotransformations catalysed by growing cells of E. coli was complicated by the production of indole in the reaction mixtures, possibly resulting from the in vivo activity of E. coli tryptophanase. Four of the recombinant strains expressing different BVMOs catalysed the oxidation of one or more of four screening substrates, well above controls that had been transformed with the re-ligated parent vector. One of the recombinant strains, E. coli B834 (DE3) pDB5, expressing the Rv3049c gene from MTb, was found to effectively resolve the target substrate, yielding a 19% yield of (1R, 5S)-(+)-bicyclo[3.2.0]hept-2-en-6-one with >95% enantiomeric excess in a 4 L fermentation reaction.