Crystallization and preliminary X-ray diffraction analysis of eukaryotic α2-macroglobulin family members modified by methylamine, proteases and glycosidases

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Summary: α2-Macroglobulin (α2M) has many functions in vertebrate physiology. To understand the basis of such functions, high-resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α2M (hα2M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα2M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X-ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well-shaped crystals routinely diffracting below 7 Å in a reproducible manner.European, Spanish, and Catalan Agencies. Grant Number: FP7-HEALTH-2010-261460; Gums&Joints. Grant Number: FP7-PEOPLE-2011-ITN-290246; RAPID. Grant Number: FP7-HEALTH-2012-306029-2; TRIGGER. Grant Numbers: BFU2012-32862, CSD2006-00015; La Marató de TV3. Grant Numbers: 2009-100732, 2009SGR1036; ESRFPeer Reviewe

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase

Theodoros Goulas et al.Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a >Michaelis loop> that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteinehistidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants.This study was financially supported in part by grants from European, US American, Polish, Spanish, and Catalan agencies (UMO-2012/04/A/NZ1/00051, UMO-2012/05/B/NZ6/00581, UMO-2013/08/W/NZ1/00696, 2137/7.PR-EU/2011/2, 2975/7.PR/13/2014/2, NIH NIDCR DE09761; FP7-PEOPLE-2011-ITN-290246 “RAPID”; FP7-HEALTH-2012-306029-2 “TRIGGER”; FP7-HEALTH-2010-261460 “Gums&Joints”; BFU2012-32862; BFU2012-33516; BFU2012-35367; BIO2013-49320-EXP; MDM-2014-0435; 2014SGR9 and 2014SGR997). IGF acknowledges an FPU-fellowship (AP2010-3799) from the former Spanish Ministry for Education, Culture and Sport. TG acknowledges a “Juan de la Cierva” research contract (JCI-2012-13573) from the Spanish Ministry for Economy and Competitiveness. The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. Funding for data collection was provided in part by ESRFPeer Reviewe

An integrative structural biology analysis of von Willebrand factor binding and processing by ADAMTS-13 in solution

Peer reviewe

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.This study was supported in part by grants from Spanish, French, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00, BES-2015-074583, ANR-10-LABX-12-01, 6108-00031B, 8022-00385B, LF18039, NNF18OC0032724, Novo Nordisk Foundation “Bio-MS”, 2017SGR3 and Fundació “La Marató de TV3” 201815). This work was also supported by EPICS-XS, project 823839, funded by the Horizon 2020 programme of the European Union. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), which are national infrastructures supported by the French National Research Agency (grants ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Finally, we acknowledge the Structural Mass Spectrometry Unit of CIISB, an Instruct-CZ Centre, which was supported by MEYS CR (LM2018127)

Structure and mechanism of a bacterial host-protein citrullinating virulence factor, Porphyromonas gingivalis peptidylarginine deiminase.

Citrullination is a post-translational modification of higher organisms that deiminates arginines in proteins and peptides. It occurs in physiological processes but also pathologies such as multiple sclerosis, fibrosis, Alzheimer's disease and rheumatoid arthritis (RA). The reaction is catalyzed by peptidylarginine deiminases (PADs), which are found in vertebrates but not in lower organisms. RA has been epidemiologically associated with periodontal disease, whose main infective agent is Porphyromonas gingivalis. Uniquely among microbes, P. gingivalis secretes a PAD, termed PPAD (Porphyromonas peptidylarginine deiminase), which is genetically unrelated to eukaryotic PADs. Here, we studied function of PPAD and its substrate-free, substrate-complex, and substrate-mimic-complex structures. It comprises a flat cylindrical catalytic domain with five-fold α/β-propeller architecture and a C-terminal immunoglobulin-like domain. The PPAD active site is a funnel located on one of the cylinder bases. It accommodates arginines from peptide substrates after major rearrangement of a "Michaelis loop" that closes the cleft. The guanidinium and carboxylate groups of substrates are tightly bound, which explains activity of PPAD against arginines at C-termini but not within peptides. Catalysis is based on a cysteine-histidine-asparagine triad, which is shared with human PAD1-PAD4 and other guanidino-group modifying enzymes. We provide a working mechanism hypothesis based on 18 structure-derived point mutants

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity

Biological and biotechnological aspects of β- and α-galactosidases from Bifidobacterium bifidum ncimb41171

In this thesis, the field of the bifidobacteria! physiology in relation to β- and α-galactosidases is described and perspectives of them for oligosaccharides synthesis are discussed- The experimental work focused on the isolation and characterisation of these cell constituents from Bifidobacterium bifidum NCIMB41171. To generate a basis for subsequent work the construction of a genomic DNA library was carried out. This resulted in the isolation of four different β-galactosidases (bbgI, bbgII, bbgIII and bbgIV) and one α-galactosidase (melA) genes. Molecular characterisation of the gene products indicated their putative location, which was subsequently confirmed after expression in an E. coli host. Nucleotide and amino acid sequence comparisons with other known bifidobacterial enzymes revealed aspects of either their evolution or conservation among species.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Biological and biotechnological aspects of β- and α-galactosidases from Bifidobacterium bifidum ncimb41171

In this thesis, the field of the bifidobacteria! physiology in relation to β- and α-galactosidases is described and perspectives of them for oligosaccharides synthesis are discussed- The experimental work focused on the isolation and characterisation of these cell constituents from Bifidobacterium bifidum NCIMB41171. To generate a basis for subsequent work the construction of a genomic DNA library was carried out. This resulted in the isolation of four different β-galactosidases (bbgI, bbgII, bbgIII and bbgIV) and one α-galactosidase (melA) genes. Molecular characterisation of the gene products indicated their putative location, which was subsequently confirmed after expression in an E. coli host. Nucleotide and amino acid sequence comparisons with other known bifidobacterial enzymes revealed aspects of either their evolution or conservation among species.EThOS - Electronic Theses Online ServiceGBUnited Kingdo