The structure of an orthorhombic crystal form of a 'forced reduced' thiol peroxidase reveals lattice formation aided by the presence of the affinity tag

Thiol peroxidase (Tpx) is an atypical 2-Cys peroxiredoxin, which has been suggested to be important for cell survival and virulence in Gram-negative pathogens. The structure of a catalytically inactive version of this protein in an orthorhombic crystal form has been determined by molecular replacement. Structural alignments revealed that Tpx is conserved. Analysis of the crystal packing shows that the linker region of the affinity tag is important for formation of the crystal lattice

A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure

Calicivirus VP2 forms a portal-like assembly following receptor engagement

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly—which was not detected in undecorated virions—is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae

Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

Escherichia coliis a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase, NapA, contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD.In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1:1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapAwas at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken altogether, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor

Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity

Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 Å resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity

Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP‐A‐containing nucleosomes. To preserve centromere identity, CENP‐A must be escorted to centromeres by a CENP‐A‐specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP‐A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X‐ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP‐A histone chaperone, binds both CENP‐A and the centromere receptor CENP‐C without the requirement for the Mis18 complex. While an N‐terminal CAL1 fragment wraps around CENP‐A/H4 through multiple physical contacts, a C‐terminal CAL1 fragment directly binds a CENP‐C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP‐A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP‐C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP‐C dimer. Overall, by demonstrating how CAL1 binds CENP‐A/H4 and CENP‐C, we provide key insights into the minimalistic principles underlying centromere maintenance

FolX from Pseudomonas aeruginosa is octameric in both crystal and solution

FolX encodes an epimerase that forms one step of the tetrahydrofolate biosynthetic pathway, which is of interest as it is an established target for important drugs. Here we report the crystal structure of FolX from the bacterial opportunistic pathogen Pseudomonas aeruginosa, as well as a detailed analysis of the protein in solution, using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). In combination, these techniques confirm that the protein is an octamer both in the crystal structure, and in solution

Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.IG is a Wellcome Senior Fellow (Ref: 207498/Z/17/Z). M.J.C. was supported by a PhD studentship from the UK Biotechnology and Biological Sciences Research Council (BBSRC WestBIO DTP: BB/J013854/1). D.B and M.M. are supported by the UK Medical Research Council (MC_UU_12014/7)

Aldehyde-alcohol dehydrogenase undergoes structural transition to form extended spirosomes for substrate channeling

Aldehyde-alcohol dehydrogenase (AdhE) is an enzyme responsible for converting acetyl-CoA to ethanol via acetaldehyde using NADH. AdhE is composed of two catalytic domains of aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH), and forms a spirosome architecture critical for AdhE activity. Here, we present the atomic resolution (3.43 Å) cryo-EM structure of AdhE spirosomes in an extended conformation. The cryo-EM structure shows that AdhE spirosomes undergo a structural transition from compact to extended forms, which may result from cofactor binding. This transition leads to access to a substrate channel between ALDH and ADH active sites. Furthermore, prevention of this structural transition by crosslinking hampers the activity of AdhE, suggesting that the structural transition is important for AdhE activity. This work provides a mechanistic understanding of the regulation mechanisms of AdhE activity via structural transition, and a platform to modulate AdhE activity for developing antibiotics and for facilitating biofuel production

Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly

The final version of this article is available at the link below.Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1–E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric ‘core’ provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an ‘average’ 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.This work was partly supported by EPSRC (under grants GR/R99393/01 and EP/C015452/1)