A dual cohesin–dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100552
ISSN1083-351X
AutoresMarlene Duarte, Aldino Viegas, Victor D. Alves, José A. M. Prates, L.M.A. Ferreira, Shabir Najmudin, Eurico J. Cabrita, Ana Luı́sa Carvalho, C.M.G.A. Fontes, Pedro Bule,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoThe Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size. The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin–dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin–dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size. Recycling of photosynthetically fixed carbon is a crucial microbial process, critical to the cycling of carbon between plants, herbivores, and microbes. Bacteroides (Pseudobacteroides) cellulosolvens is a mesophilic, anaerobic bacterium capable of degrading crystalline cellulose (1Giuliano C. Khan A.W. Cellulase and sugar formation by Bacteroides cellulosolvens, a newly isolated cellulolytic anaerobe.Appl. Environ. Microbiol. 1984; 48: 446-448Crossref PubMed Google Scholar, 2Giuliano C. Khan A.W. Conversion of cellulose to sugars by resting cells of a mesophilic anaerobe, Bacteriodes cellulosolvens.Biotechnol. Bioeng. 1985; 27: 980-983Crossref PubMed Scopus (16) Google Scholar). Similar to other bacteria such as Clostridium (Hungateiclostridium) thermocellum and Acetivibrio (Hungateiclostridium) cellulolyticus, Bacteroides cellulosolvens produces an extracellular multimodular cellulolytic complex—the cellulosome—responsible for the degradation of the plant cell wall (3Ding S.-Y. Bayer E.A. Steiner D. Shoham Y. Lamed R. A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins.J. Bacteriol. 2000; 182: 4915-4925Crossref PubMed Scopus (59) Google Scholar). Noteworthy, B. cellulosolvens has the most intricate cellulosome so far described, conceivably capable of congregating up to 110 carbohydrate-active enzymes in a cell-associated mega-Dalton complex (Fig. 1) (4Zhivin O. Dassa B. Moraïs S. Utturkar S.M. Brown S.D. Henrissat B. Lamed R. Bayer E.A. Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system.Biotechnol. Biofuels. 2017; 10: 211Crossref PubMed Scopus (22) Google Scholar). Cellulosomes are built around a primary noncatalytic protein scaffold, named scaffoldin, bearing reiterated cohesin (Coh) modules that serve as protein–protein interaction targets to dockerin (Doc) modules found in an extensive repertoire of independent Carbohydrate-Active enZymes (CAZymes). The ability to gather a large number of diverse enzymes into the cellulosome presumably provides these anaerobic microorganisms an important advantage in their competitive ecological niches, through complementary and synergic enzyme cooperation, while also promoting enzyme stability (5Fontes C.M.G.A. Gilbert H.J. Cellulosomes: Highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates.Annu. Rev. Biochem. 2010; 79: 655-681Crossref PubMed Scopus (410) Google Scholar). The cellulosome's Coh-Doc protein:protein interactions constitute the primary driving force for cellulosomal assembly and are among nature's strongest protein:protein interactions (Ka > 109 M−1). Also, due to a distinctive twofold internal symmetry, Doc modules can potentially bind their cognate Cohs in two different orientations, by rotating 180° with respect to its protein ligand (6Carvalho A.L. Dias F.M.V. Nagy T. Prates J.A.M. Proctor M.R. Smith N. Bayer E.A. Davies G.J. Ferreira L.M.A. Romão M.J. Fontes C.M.G.A. Gilbert H.J. Evidence for a dual binding mode of dockerin modules to cohesins.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3089-3094Crossref PubMed Scopus (106) Google Scholar). This is known as a dual-binding mode, as opposed to a single-binding mode that occurs when only one of the Doc interfaces supports the formation of the Coh-Doc complex (7Adams J.J. Webb B.A. Spencer H.L. Smith S.P. Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: Calcium-induced effects on conformation and target recognition †.Biochemistry. 2005; 44: 2173-2182Crossref PubMed Scopus (34) Google Scholar). A sequence-based classification of Cohs and Docs distinguishes type I and type II interactions as major categories (8Brás J.L.A. Pinheiro B.A. Cameron K. Cuskin F. Viegas A. Najmudin S. Bule P. Pires V.M.R. Romão M.J. Bayer E.A. Spencer H.L. Smith S. Gilbert H.J. Alves V.D. Carvalho A.L. et al.Diverse specificity of cellulosome attachment to the bacterial cell surface.Sci. Rep. 2016; 6: 38292Crossref PubMed Scopus (17) Google Scholar). In the archetypal cellulosome of Clostridium thermocellum, the assembly of the different enzymes into the main scaffoldins is mediated by type I Coh-Doc interactions with a dual-binding mode, whereas the anchoring of the scaffoldin to the bacterial cell wall is achieved through type II single-binding mode Coh-Doc interactions (5Fontes C.M.G.A. Gilbert H.J. Cellulosomes: Highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates.Annu. Rev. Biochem. 2010; 79: 655-681Crossref PubMed Scopus (410) Google Scholar, 9Bayer E.A. Belaich J.P. Shoham Y. Lamed R. The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides.Annu. Rev. Microbiol. 2004; 58: 521-554Crossref PubMed Scopus (717) Google Scholar). Although there are noteworthy exceptions, such as a generalized Coh-Doc dual-binding mode in the cellulosome of Acetivibrio cellulolyticus (8Brás J.L.A. Pinheiro B.A. Cameron K. Cuskin F. Viegas A. Najmudin S. Bule P. Pires V.M.R. Romão M.J. Bayer E.A. Spencer H.L. Smith S. Gilbert H.J. Alves V.D. Carvalho A.L. et al.Diverse specificity of cellulosome attachment to the bacterial cell surface.Sci. Rep. 2016; 6: 38292Crossref PubMed Scopus (17) Google Scholar, 10Cameron K. Najmudin S. Alves V.D. Bayer E.A. Smith S.P. Bule P. Waller H. Ferreira L.M.A. Gilbert H.J. Fontes C.M.G.A. Cell-surface attachment of bacterial multienzyme complexes involves highly dynamic protein-protein anchors.J. Biol. Chem. 2015; 290: 13578-13590Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar) or, conversely, a ubiquitous single-binding mode found in Ruminococcus flavefaciens (11Bule P. Alves V.D. Leitão A. Ferreira L.M.A. Bayer E.A. Smith S.P. Gilbert H.J. Najmudin S. Fontes C.M.G.A. Single-binding mode integration of hemicellulose degrading enzymes via adaptor scaffoldins in Ruminococcus flavefaciens cellulosome.J. Biol. Chem. 2016; 291: 26658-26669Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), this has been considered the rule. Based on the almost complete genome sequence of B. cellulosolvens (12Dassa B. Utturkar S. Hurt R.A. Klingeman D.M. Keller M. Xu J. Reddy Y.H.K. Borovok I. Rozman Grinberg I. Lamed R. Zhivin O. Bayer E.A. Brown S.D. Near-complete genome sequence of the cellulolytic bacterium Bacteroides (Pseudobacteroides) cellulosolvens ATCC 35603.Genome Announc. 2015; 3e01022-15Crossref PubMed Scopus (8) Google Scholar), Zhivin et al. (4Zhivin O. Dassa B. Moraïs S. Utturkar S.M. Brown S.D. Henrissat B. Lamed R. Bayer E.A. Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system.Biotechnol. Biofuels. 2017; 10: 211Crossref PubMed Scopus (22) Google Scholar) performed a seminal study on the architecture and functional organization of the most complex cellulosome described so far. Their work has identified 31 different scaffoldins, many of which lack any known cell-surface binding domains, thus supporting an extensive putative cell-free cellulosome system. Besides a novel classification of Coh-Doc pairings, named type R, another striking feature of this cellulosome is a reversal of Coh types found in scaffoldins. Unlike in other species, B. cellulosolvens'primary scaffoldin recruitment of Doc-bearing enzymes is mediated by type II Coh-Doc interactions, while anchoring scaffoldins rely on type I Coh-Doc complexes for cell wall attachment. The primary scaffoldin subunit of B. cellulosolvens, termed ScaA1, like other primary scaffoldin proteins, incorporates a carbohydrate-binding module (family 3 CBM), but is unusually composed of 11 type II Cohs and a C-terminal type I Doc that does not have an associated X module, which is a known type II Coh-Doc interaction stabilizer (3Ding S.-Y. Bayer E.A. Steiner D. Shoham Y. Lamed R. A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins.J. Bacteriol. 2000; 182: 4915-4925Crossref PubMed Scopus (59) Google Scholar). Cell-surface anchoring of ScaA1 occurs via interaction of its single type I Doc with one of the ten type I Cohs found in the ScaB anchoring scaffoldin. Anchoring to the peptidoglycan-associated polymers from the bacterium cell surface likely results from a noncovalent interaction with ScaB's S-layer homology (SLH) domain (13Zhao G. Li H. Wamalwa B. Sakka M. Kimura T. Sakka K. Different binding specificities of S-layer homology modules from Clostridium thermocellum AncA, Slp1, and Slp2.Biosci. Biotechnol. Biochem. 2006; 70: 1636-1641Crossref PubMed Scopus (12) Google Scholar). This SLH-mediated interaction is likely aided by an adjacent X module, another B. cellulosolvens peculiarity (4Zhivin O. Dassa B. Moraïs S. Utturkar S.M. Brown S.D. Henrissat B. Lamed R. Bayer E.A. Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system.Biotechnol. Biofuels. 2017; 10: 211Crossref PubMed Scopus (22) Google Scholar). While the functional implications of the reversed specificity of Coh types in B. cellulosolvens cellulosome architecture remain unclear, the actual structural determinants of specificity of its Coh-Doc type II interactions are also currently unknown. This knowledge gap is relevant considering that, although known cellulosome Coh-Doc structures share a remarkable overall conservation of structural topology, the major determinants for interspecies and intraspecies barriers, as well as for the binding mode, depend on very subtle amino acid residue differences (14Bule P. Cameron K. Prates J.A.M. Ferreira L.M.A. Smith S.P. Gilbert H.J. Bayer E.A. Najmudin S. Fontes C.M.G.A. Alves V.D. Structure–function analyses generate novel specificities to assemble the components of multi-enzyme bacterial cellulosome complexes.J. Biol. Chem. 2018; 293: 4201-4212Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The structure of B. cellulosolvens isolated 11th type II Coh module of BcScaA1 (PDB code: 1tyj (15Noach I. Frolow F. Jakoby H. Rosenheck S. Shimon L.W. Lamed R. Bayer E.A. Crystal structure of a type-II cohesin module from the Bacteroides cellulosolvens cellulosome reveals novel and distinctive secondary structural elements.J. Mol. Biol. 2005; 348: 1-12Crossref PubMed Scopus (26) Google Scholar)), BcScaA1-CBM3 (PDB code: 2xbt, (16Yaniv O. Shimon L.J.W. Bayer E.A. Lamed R. Frolow F. Scaffoldin-borne family 3b carbohydrate-binding module from the cellulosome of Bacteroides cellulosolvens: Structural diversity and significance of calcium for carbohydrate binding.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 506-515Crossref PubMed Scopus (16) Google Scholar)), and seventh type I Coh module of BcScaB (PDB code: 4ums, (10Cameron K. Najmudin S. Alves V.D. Bayer E.A. Smith S.P. Bule P. Waller H. Ferreira L.M.A. Gilbert H.J. Fontes C.M.G.A. Cell-surface attachment of bacterial multienzyme complexes involves highly dynamic protein-protein anchors.J. Biol. Chem. 2015; 290: 13578-13590Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar)) were previously reported. The latter report suggested a dual-binding mode for B. cellulosolvens cellulosomal cell anchoring. This presumed plasticity for cell anchoring binding mode is similar to that found on A. cellulolyticus Coh-Doc interactions involving the primary scaffoldin (AcScaA), a unique adaptor scaffoldin (AcScaB) and several anchoring scaffoldins (AcScaC, AcScaD, and AcScaF) (PDB codes 4u3s/4wi0 (8Brás J.L.A. Pinheiro B.A. Cameron K. Cuskin F. Viegas A. Najmudin S. Bule P. Pires V.M.R. Romão M.J. Bayer E.A. Spencer H.L. Smith S. Gilbert H.J. Alves V.D. Carvalho A.L. et al.Diverse specificity of cellulosome attachment to the bacterial cell surface.Sci. Rep. 2016; 6: 38292Crossref PubMed Scopus (17) Google Scholar), and 4uyp/4uyq, (10Cameron K. Najmudin S. Alves V.D. Bayer E.A. Smith S.P. Bule P. Waller H. Ferreira L.M.A. Gilbert H.J. Fontes C.M.G.A. Cell-surface attachment of bacterial multienzyme complexes involves highly dynamic protein-protein anchors.J. Biol. Chem. 2015; 290: 13578-13590Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). All of these are in contradiction to the canonical single-binding mode found for cellulosome cell anchoring in C. thermocellum (PDB code 2b59, (17Adams J.J. Pal G. Jia Z. Smith S.P. Mechanism of bacterial cell-surface attachment revealed by the structure of cellulosomal type II cohesin-dockerin complex.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 305-310Crossref PubMed Scopus (84) Google Scholar). The 11th type II Coh structure of BcScaA1 shows an overall fold similarity with several type II Cohs from A. cellulolyticus, namely with AcCohScaB3 (PDB code 4u3s), with a Q-score of 0.79 and root-mean-square deviation (rmsd) of 1.22 Å over 163 aligned Cα residues. It also bears close homology to the type II CtCohScaF (PDB code 2b59) with a Q-score of 0.77 and rmsd of 1.40 Å against 164 aligned Cα residues. They all share the characteristic α-helical crowning between strands 6 and 7 and the two singular β-flaps that disrupt strands 4 and, particularly, strand 8 where its 12 residues are flanking the type II Coh Doc-binding plateau. The elucidation of B. cellulosolvens cellulosome assembly and the reversal of Coh types found in its scaffoldins hinge upon the availability of type II CohScaA-Doc complex structures to understand the consequences of this unusual arrangement. Here is reported the crystal structure of the type II 11th Coh of the primary scaffoldin of B. cellulosolvens in complex with the Doc module of a glycoside hydrolase of family 48, BcCohScaA111-DocCel48 (PDB code: 2y3n). A detailed binding characterization informed by the structural data has also been carried out, which allowed the identification of the molecular determinants of Coh-Doc interaction and suggested a typical dual-binding mode for cellulosome enzyme assembly, thus agreeing with the common paradigm for a flexible arrangement, as found on C. thermocellum (6Carvalho A.L. Dias F.M.V. Nagy T. Prates J.A.M. Proctor M.R. Smith N. Bayer E.A. Davies G.J. Ferreira L.M.A. Romão M.J. Fontes C.M.G.A. Gilbert H.J. Evidence for a dual binding mode of dockerin modules to cohesins.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 3089-3094Crossref PubMed Scopus (106) Google Scholar). In light of this report and considering the putative dual-binding mode for scaffoldin assembly in both B. cellulosolvens and A. cellulolyticus (10Cameron K. Najmudin S. Alves V.D. Bayer E.A. Smith S.P. Bule P. Waller H. Ferreira L.M.A. Gilbert H.J. Fontes C.M.G.A. Cell-surface attachment of bacterial multienzyme complexes involves highly dynamic protein-protein anchors.J. Biol. Chem. 2015; 290: 13578-13590Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), it is fair to conclude that the single versus dual-binding mode in Coh-Doc complexes is independent of type classification. It also does not seem to be a matter of enzyme assembly versus cell-anchoring/scaffoldin assembly. Rather, it might be related to the size and complexity that is possible to achieve in a single unit within a cellulosomal system, with larger cellulosomes requiring a higher degree of flexibility for proper assembly and access to substrate. A critical factor to understand the mechanism of cellulosome assembly of B. cellulosolvens is the availability of an X-ray crystal structure of the type II Coh-Doc interaction, central to CAZyme assembly around the primary BcScaA1 scaffoldin. Escherichia coli coexpression strategies for the production and purification of Coh-Doc complexes were thus used to obtain good-quality crystals of highly pure protein complexes of the type II 11th Coh of the primary scaffoldin of B. cellulosolvens (BcCohScaA111), in complex with the Doc module of a glycoside hydrolase of family 48 (DocCel48). A previous study described the structure of this Coh in its unbound form (PDB code: 1tyj (15Noach I. Frolow F. Jakoby H. Rosenheck S. Shimon L.W. Lamed R. Bayer E.A. Crystal structure of a type-II cohesin module from the Bacteroides cellulosolvens cellulosome reveals novel and distinctive secondary structural elements.J. Mol. Biol. 2005; 348: 1-12Crossref PubMed Scopus (26) Google Scholar)), and solving its structure in complex with a bound Doc would allow probing structural differences arising from Coh binding to a Doc partner. The chosen Doc module belongs to one of the most abundant cellulosomal CAZymes, GH48 cellobiohydrolase, and was previously reported to bind Cohs from ScaA1 (4Zhivin O. Dassa B. Moraïs S. Utturkar S.M. Brown S.D. Henrissat B. Lamed R. Bayer E.A. Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system.Biotechnol. Biofuels. 2017; 10: 211Crossref PubMed Scopus (22) Google Scholar, 18Haimovitz R. Barak Y. Morag E. Voronov-Goldman M. Shoham Y. Lamed R. Bayer E.A. Cohesin-dockerin microarray: Diverse specificities between two complementary families of interacting protein modules.Proteomics. 2008; 8: 968-979Crossref PubMed Scopus (83) Google Scholar). The high degree of sequence conservation between the DocCel48's two dockerin repeats suggests the existence of two cohesin-binding interfaces, thus supporting a dual-binding mode. This implies that two different complex conformations could be present in solution, which would likely compromise protein crystallization due to a lack of unit cell homogeneity. It is well established that residues at relative positions 10 and 11 of each of the two Doc duplicated segments play a key role in Coh recognition and act as specificity determinants (residues #17, #18, #50, and #51 of the construct used in this work) (9Bayer E.A. Belaich J.P. Shoham Y. Lamed R. The cellulosomes: Multienzyme machines for degradation of plant cell wall polysaccharides.Annu. Rev. Microbiol. 2004; 58: 521-554Crossref PubMed Scopus (717) Google Scholar). Thus, a DocCel48 mutant was designed to force binding through a single interface, promoting homogeneity of the purified protein. The mutations used for the crystallization experiments were designed to replace the putative recognition residues in relative positions 10 and 11 of the C-terminal Doc repeat (Met50 and Ala51) with those of the B. cellulosolvens ScaA type I Doc (Ser-Asp), rather than the commonly applied alanine substitution. These amino acid changes were chosen based on the lack of cross-reaction between type I and type II Coh-Doc complexes. The sequence of the resulting Doc is displayed in Table S2. This strategy allowed us to obtain large yields of highly pure Coh-Doc complexes for crystallization, which led to the production of well-diffracting crystals. A molecular replacement strategy was used to solve the complex's structure, using the available BcCohScaA111 structure as an input model (PDB code: 1tyj (15Noach I. Frolow F. Jakoby H. Rosenheck S. Shimon L.W. Lamed R. Bayer E.A. Crystal structure of a type-II cohesin module from the Bacteroides cellulosolvens cellulosome reveals novel and distinctive secondary structural elements.J. Mol. Biol. 2005; 348: 1-12Crossref PubMed Scopus (26) Google Scholar)). This yielded a solution with two Cohs in the asymmetric unit. Successive rounds of automated ARP/wARP (19Langer G. Cohen S.X. Lamzin V.S. Perrakis A. Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7.Nat. Protoc. 2008; 3: 1171-1179Crossref PubMed Scopus (1295) Google Scholar) and manual COOT (20Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (16100) Google Scholar) adjustments to build the Doc modules in both crystallographic Coh-Doc dimer complexes resulted in a final REFMAC5 (21Murshudov G.N. Skubák P. Lebedev A.A. Pannu N.S. Steiner R.A. Nicholls R.A. Winn M.D. Long F. Vagin A.A. REFMAC 5 for the refinement of macromolecular crystal structures.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 355-367Crossref PubMed Scopus (5716) Google Scholar) refined structure at 1.90 Å resolution. The two molecules of the BcCohScaA111-DocCel48 heterodimer share 299 water molecules and each Doc is coordinating two calcium (Ca2+) ions. The Coh-Doc complex includes residues 2073 to 2242 from BcCohScaA111 (Nonredundant RefSeq accession number AAG01230), 683 to 752 from BcDocCel48A (Nonredundant RefSeq accession number WP_050753099). The structure belongs to the monoclinic space group P1211 with unit cell dimensions of a = 43.4 Å, b = 116.1 Å, c = 45.2 Å, and β = 112.5°. Due to disorder, some of the dockerin's helix 2 residues could not be modeled, namely those between Gly32 and Asn37 of chain B and 15 residues from Ala30 to Asn44 in chain D. Some C- and N-terminal residues, including the 6-histidine tag, are also absent in all four chains. The final model was deposited in the Protein Data Bank under accession code 2y3n. Data collection and refinement statistics are shown in Table 1.Table 1X-ray crystallography data collection and refinement statistics for BcCohScaA1-DocCel48Data qualityBcCohScaA1-DocCel48Cell dimensions, Åa = 43.4b = 116.1c = 45.2β = 112.5°Space groupP1211X-ray sourceESRF, ID14-EH1Wavelength, Å0.934Resolution of data (outer shell), Å41.74–1.90 (2.00–1.90)Rpim (outer shell)aRmerge=∑hkl∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), where I is the observed intensity, and I¯ is the statistically-weighted average intensity of multiple observations. Rp.i.m.=∑hkl1/(n−1)∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), a redundancy-independent version of Rmerge.0.073 (0.278)Rmerge (outer shell)aRmerge=∑hkl∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), where I is the observed intensity, and I¯ is the statistically-weighted average intensity of multiple observations. Rp.i.m.=∑hkl1/(n−1)∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), a redundancy-independent version of Rmerge.0.090–0.051 (0.329)Mean I/σ (I) (outer shell)15.0 (3.9)Completeness (outer shell), %83.9 (66.4)Multiplicity (outer shell)2.40 (2.2)Structure quality N° of protein atoms (AU)3765 N° calcium atoms4 N° solvent waters299 Resolution used in refinement, Å1.90 Rwork/Rfree,%bRwork=∑hkl||Fobs(hkl)|−|Fcalc(hkl)||∑hkl|Fobs(hkl)|, where |Fcalc| and |Fobs| are the calculated and observed structure factor amplitudes, respectively. (Rfree is calculated for a randomly chosen 5% of the reflections).16.3/22.5Average temperature factors, Å2 Main chain (CohA, DocB, CohC, DocD)21.3, 31.0, 21.2, 44.3 Side chain (CohA, DocB, CohC, DocD)24.4, 33.0, 24.2, 47.7 Calcium atoms (B1, B2, D1, D2)25.6, 22.9, 27.3, 61.84 Solvent waters39.3RMS deviations Bond lengths, Å0.022 Bond angles, °1.696Ramachandran's plot analysis Favorable, %96.1 Allowed, %3.6 Outlier, %0.2PDB accession code2y3nValues in parenthesis are for the highest resolution shell.a Rmerge=∑hkl∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), where I is the observed intensity, and I¯ is the statistically-weighted average intensity of multiple observations. Rp.i.m.=∑hkl1/(n−1)∑i=1n|Ii(hkl)−I¯(hkl)|∑hkl∑i=1nIi(hkl), a redundancy-independent version of Rmerge.b Rwork=∑hkl||Fobs(hkl)|−|Fcalc(hkl)||∑hkl|Fobs(hkl)|, where |Fcalc| and |Fobs| are the calculated and observed structure factor amplitudes, respectively. (Rfree is calculated for a randomly chosen 5% of the reflections). Open table in a new tab Values in parenthesis are for the highest resolution shell. The cohesin domain of the type II Coh11-Doc complex of B. cellulosolvens shows the typical flattened, elongated 9-stranded β-barrel jelly-roll topology (Fig. 2A) with a highly hydrophobic core. Similar to the C. thermocellum structure, the nine β-strands define two β-sheets—the first β-sheet is defined by strands 8-3-6-5 (front face) and the second is defined by strands 9-1-2-7 (back face). The common α-helical crowning observed between strands 6 and 7 and the two β-flap regions that disrupt the normal progression of strands 4 and 8 are maintained (10Cameron K. Najmudin S. Alves V.D. Bayer E.A. Smith S.P. Bule P. Waller H. Ferreira L.M.A. Gilbert H.J. Fontes C.M.G.A. Cell-surface attachment of bacterial multienzyme complexes involves highly dynamic protein-protein anchors.J. Biol. Chem. 2015; 290: 13578-13590Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 11Bule P. Alves V.D. Leitão A. Ferreira L.M.A. Bayer E.A. Smith S.P. Gilbert H.J. Najmudin S. Fontes C.M.G.A. Single-binding mode integration of hemicellulose degrading enzymes via adaptor scaffoldins in Ruminococcus flavefaciens cellulosome.J. Biol. Chem. 2016; 291: 26658-26669Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 15Noach I. Frolow F. Jakoby H. Rosenheck S. Shimon L.W. Lamed R. Bayer E.A. Crystal structure of a type-II cohesin module from the Bacteroides cellulosolvens cellulosome reveals novel and distinctive secondary structural elements.J. Mol. Biol. 2005; 348: 1-12Crossref PubMed Scopus (26) Google Scholar), with the latter making further contact with the dockerin counterpart. Comparing this structure with that of the unbound BcCohScaA111 (1tyj) shows that, globally, the Coh does not undergo significant conformational changes upon binding, as revealed by the low rmsd value (0.66 Å for 166 Cα atoms) between both structures. Nonetheless, some differences can be found on the binding plateau that better accommodates the ligand partner, namely on the β-strand 8 loop (defined by residues 136 and 145) as indicated by a larger rmsd value of 1.35 Å between 11 atom pairs. The closest BcCohScaA111 functionally relevant structural homologues according to the PDBeFold server (http://www.ebi.ac.uk/msd-srv/ssm/) are type II cohesins from A. cellulolyticus ScaB (PDB accession codes 3bwz and 4u3s), responsible for the anchoring between adaptor scaffoldin ScaB and main scaffoldin ScaA through a dual-binding mode interaction. These protein modules were matched with a Z-score of 11.4, rmsd of 1.18 Å, sequence identity of 44% over 163 aligned residues and Z-score of 12.6, rmsd of 1.22 Å, sequence identity of 40% over 162 aligned residues, respectively. The type II cohesin from C. thermocellum's ScaF (SdbA) also shares a high degree of structural
Referência(s)