Serial macromolecular crystallography at ALBA Synchrotron Light Source

12 pags., 4 figs., 2 tabs. -- Addenda and errata: https://journals.iucr.org/s/issues/2022/03/00/rv5160/rv5160.pdfThe increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 μm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 μm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.The following funding is acknowledged: Ayuda de Atracciony Retencion de Talento Investigador" from the Community of Madrid (scholarship No. 2019-T1/BMD-15552); STC Programof the National Science Foundation through BioXFEL (awardNo. 1231306); the Centre for Applied Structural Discovery(CASD) at the Biodesign Institute at Arizona State University; the Spanish Ministry of Science and Innovation, grants EQC2021-007532-P, PID2020-117028GB-I00, BIO2016-77883-C2-2-

Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo

Biochemical and Structural Characterization of Mycoplasma genitalium proteins MG438 and MG20

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The race to resolve the atomic structures of the ribosome. On the Nobel Prize in Chemistry awarded to Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath

The Nobel Prize in Chemistry 2009 was awarded to three scientists, Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath, for their investigations into the structure and functioning of ribosomes. These complex cellular particles are where genetic information is decoded and proteins are synthesized. Consequently, ribosomes play a central role inthe biology of all living organisms. Ribosomes are composed of one small and one large subunit, which in prokaryotes are respectively referred to as 30S and 50S according to their sedimentation properties. In both subunits, about two thirds of the mass corresponds to ribosomal RNA (rRNA) and the rest to different proteins. Given their biological relevance and the fact that they are the target of a large variety of clinically relevant antibiotics, ribosomes have been the subject of intense and continuousresearch since the 1960s, when the genetic code was unraveled. These investigations led, and to some extent culminated, with the results published in 2000 (annus mirabilis for ribosomes), reporting the crystal structures of the 50S ribosomal subunit from Haloarcula marismortui at 2.4Å resolution and, a few weeks later, of the 30S subunit from Thermus thermophilus at 3.3Å and 3.0Å resolution, by teams led by the three laureates. These results have been instrumental in understanding ribosome function at the atomic level. However, there are many years of work ahead, as much remains to be learned about ribosomes; in particular the structure of the eukaryotic ribosome has yet to be elucidated

Venkatraman Ramakrishnan, Thomas A. Steitz y Ada E. Yonath. Premios Nobel de Química 2009: "por sus estudios sobre la estructura y funcion del Ribosoma"

The Nobel Prize in Chemistry 2009 has been awarded to Venkatraman Ramakrishnan (born in Tamil Nadu �{India�{�nin 1952) presently at the MRC Laboratory of Molecular Biology in Cambridge (United Kingdom), to Thomas A. Steitz (born in Milwaukee �{WI, USA-�nin 1940) Sterling Professor at Yale University and to Ada E. Yonath (born in Jerusalem �{Israel-�nin 1939) at the Weizmann Institute of Science, for their investigations on the structure and functioning of the ribosome. The ribosomes are complex cellular particles where takes place the decoding of the genetic information and the synthesis of proteins and, consequently, play a central role in the biology of all living organisms.El premio Nobel de Quimica del 2009 ha sido concedido a Venkatraman Ramakrishnan (nacido en Tamil Nadu ¡VIndia-�nen 1952) actualmente en el MRC Laboratory of Molecular Biology de Cambridge (Reino Unido), a Thomas A. Steitz (nacido en Milwaukee �{WI, USA-�nen 1940) Sterling Professor en Yale University y a Ada E. Yonath (nacida en Jerusalen �{Israel-�nen 1939) del Weizmann Institute of Science, por sus investigaciones sobre la estructura y funcion del ribosoma. Los ribosomas son particulas celulares complejas en las que tiene lugar la decodificacion de la informacion genetica y la sintesis de proteinas y juegan, por consiguiente, un papel central en la biologia de todos los seres vivos

The nucleotide‐bound/substrate‐bound conformation of the Mycoplasma genitalium DnaK chaperone

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms tochanges in external and internal conditions. Hsp70s have two domains—nucleotide bindingdomain (NBD) and substrate binding domain (SBD)—connected by a conserved hydrophobiclinker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allostericallycoupled, often together with cochaperones, to the binding/release of peptide substrates. Here wedescribe the crystal structure of theMycoplasma genitaliumDnaK (MgDnaK) protein, an Hsp70homolog, in the noncompact, nucleotide-bound/substrate-bound conformation. TheMgDnaKstructure resembles the one from the thermophilic eubacteria DnaK trapped in the same state.However, inMgDnaK the NBD and SBD domains remain close to each other despite the lack ofdirect interaction between them and with the linker contacting the two subdomains of SBD.These observations suggest that the structures might represent an intermediate of the proteinwhere the conserved linker binds to the SBD to favor the noncompact state of the protein by sta-bilizing the SBDb-SBDasubdomains interaction, promoting the capacity of the protein to sampledifferent conformations, which is critical for proper functioning of the molecular chaperone allo-steric mechanism.Comparison of the solved structures indicates that the NBD remains essentially invariant inpresence or absence of nucleotide.Ministerio Economía y Competitividad . Grant Number: BFU2015‐71092‐PPeer reviewe

Crystal structure of Brucella abortus deoxyxylulose-5- phosphate reductoisomerase-like (DRL) enzyme involved in isoprenoid biosynthesis

Most bacteria use the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-D-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was supported by a FPI fellowship (to J. P. G.), Research Grants BFU2009-09268 (to I. F.) and BIO2008-00432 (to M. R.-C.), and Consolider Program Grant CSD2007-00036 from the Spanish Ministerio de Ciencia e Innovación and by Grants 2009SGR-26 and XRB from the Generalitat de Catalunya.Peer Reviewe

The EAGR box structure: a motif involved in mycoplasma motility

Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self-replicating organisms. M.genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell-host adhesion. A well-conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M.genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra-subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra- and inter-subunit symmetry axes, support a role for the EAGR box in protein-protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility. © 2012 Blackwell Publishing Ltd.Funded by: Ministerio de Ciencia e Innovación. Grant Numbers: BFU2009-09268, BIO2007-67904-C02-01, BFU2010-22209-C02-01.Peer Reviewe

Structure-guided mutations in the terminal organelle protein MG491 cause major motility and morphologic alterations on mycoplasma genitalium

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle

Structure-Guided Mutations in the Terminal Organelle Protein MG491 Cause Major Motility and Morphologic Alterations on Mycoplasma genitalium

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organell