Homologous Ad26.COV2.S vaccination results in reduced boosting of humoral responses in hybrid immunity, but elicits antibodies of similar magnitude regardless of prior infection.

The impact of previous SARS-CoV-2 infection on the durability of Ad26.COV2.S vaccine-elicited responses, and the effect of homologous boosting has not been well explored. We followed a cohort of healthcare workers for 6 months after receiving the Ad26.COV2.S vaccine and a further one month after they received an Ad26.COV2.S booster dose. We assessed longitudinal spike-specific antibody and T cell responses in individuals who had never had SARS-CoV-2 infection, compared to those who were infected with either the D614G or Beta variants prior to vaccination. Antibody and T cell responses elicited by the primary dose were durable against several variants of concern over the 6 month follow-up period, regardless of infection history. However, at 6 months after first vaccination, antibody binding, neutralization and ADCC were as much as 59-fold higher in individuals with hybrid immunity compared to those with no prior infection. Antibody cross-reactivity profiles of the previously infected groups were similar at 6 months, unlike at earlier time points, suggesting that the effect of immune imprinting diminishes by 6 months. Importantly, an Ad26.COV2.S booster dose increased the magnitude of the antibody response in individuals with no prior infection to similar levels as those with previous infection. The magnitude of spike T cell responses and proportion of T cell responders remained stable after homologous boosting, concomitant with a significant increase in long-lived early differentiated CD4 memory T cells. Thus, these data highlight that multiple antigen exposures, whether through infection and vaccination or vaccination alone, result in similar boosts after Ad26.COV2.S vaccination.

The Poly-Arginine Peptide R18D Interferes with the Internalisation of α-Synuclein Pre-Formed Fibrils in STC-1 Enteroendocrine Cells.

In Parkinson's disease (PD), gut inflammation is hypothesised to contribute to α-synuclein aggregation, but gastrointestinal α-synuclein expression is poorly characterised. Cationic arginine-rich peptides (CARPs) are an emerging therapeutic option that exerts various neuroprotective effects and may target the transmission of protein aggregates. This study aimed to investigate endogenous α-synuclein expression in enteroendocrine STC-1 cells and the potential of the CARP, R18D (18-mer of D-arginine), to prevent internalisation of pre-formed α-synuclein fibrils (PFFs) in enteroendocrine cells in vitro. Through confocal microscopy, the immunoreactivity of full-length α-synuclein and the serine-129 phosphorylated form (pS129) was investigated in STC-1 (mouse enteroendocrine) cells. Thereafter, STC-1 cells were exposed to PFFs tagged with Alexa-Fluor 488 (PFF-488) for 2 and 24 h and R18D-FITC for 10 min. After confirming the uptake of both PFFs and R18D-FITC through fluorescent microscopy, STC-1 cells were pre-treated with R18D (5 or 10 µM) for 10 min prior to 2 h of PFF-488 exposure. Immunoreactivity for endogenous α-synuclein and pS129 was evident in STC-1 cells, with prominent pS129 staining along cytoplasmic processes and in perinuclear areas. STC-1 cells internalised PFFs, confirmed through co-localisation of PFF-488 and human-specific α-synuclein immunoreactivity. R18D-FITC entered STC-1 cells within 10 min and pre-treatment of STC-1 cells with R18D interfered with PFF uptake. The endogenous presence of α-synuclein in enteroendocrine cells, coupled with their rapid uptake of PFFs, demonstrates a potential for pathogenic spread of α-synuclein aggregates in the gut. R18D is a novel therapeutic approach to reduce the intercellular transmission of α-synuclein pathology.

Homologous Ad26.COV2.S vaccination results in reduced boosting of humoral responses in hybrid immunity, but elicits antibodies of similar magnitude regardless of prior infection.

The impact of previous SARS-CoV-2 infection on the durability of Ad26.COV2.S vaccine-elicited responses, and the effect of homologous boosting has not been well explored. We followed a cohort of healthcare workers for 6 months after receiving the Ad26.COV2.S vaccine and a further one month after they received an Ad26.COV2.S booster dose. We assessed longitudinal spike-specific antibody and T cell responses in individuals who had never had SARS-CoV-2 infection, compared to those who were infected with either the D614G or Beta variants prior to vaccination. Antibody and T cell responses elicited by the primary dose were durable against several variants of concern over the 6 month follow-up period, regardless of infection history. However, at 6 months after first vaccination, antibody binding, neutralization and ADCC were as much as 33-fold higher in individuals with hybrid immunity compared to those with no prior infection. Antibody cross-reactivity profiles of the previously infected groups were similar at 6 months, unlike at earlier time points suggesting that the effect of immune imprinting diminishes by 6 months. Importantly, an Ad26.COV2.S booster dose increased the magnitude of the antibody response in individuals with no prior infection to similar levels as those with previous infection. The magnitude of spike T cell responses and proportion of T cell responders remained stable after homologous boosting, concomitant with a significant increase in long-lived early differentiated CD4 memory T cells. Thus, these data highlight that multiple antigen exposures, whether through infection and vaccination or vaccination alone, result in similar boosts after Ad26.COV2.S vaccination.

An updated interactive database for 1692 genetic variants in coagulation factor IX provides detailed insights into hemophilia B.

BACKGROUND: Genetic variants in coagulation factor IX (FIX) are associated with hemophilia B, a rare bleeding disease. F9 variants are widespread across the gene and were summarized in our FIX variant database introduced in 2013. OBJECTIVES: We aimed to rationalize the molecular basis for 598 new F9 variants and 1645 new clinical cases, totaling 1692 F9 variants and 5358 related patient cases. METHODS: New F9 variants were identified from publications and online resources, and compiled into a MySQL database for comparison with the human FIXa protein structure. RESULTS: The new total of 1692 F9 variants correspond to 406 (88%) of the 461 FIX residues and now include 70 additional residues. They comprise 945 unique point variants, 281 deletions, 352 polymorphisms, 63 insertions, and 51 others. Most FIX variants were point variants, although their proportion (56%) has reduced compared to 2013 (73%); at the same time, the proportion of polymorphisms has increased from 5% to 21%. The 764 unique mild severity variants in the mature protein with known phenotypes include 74 (9.7%) quantitative type I variants and 116 (15.2%) predominantly qualitative type II variants. The remaining 574 variants types are unspecified. Inhibitors are associated with 152 hemophilia B cases out of 5358 patients (2.8%), an increase of 93 from the previous database. CONCLUSION: The even distribution of the F9 variants revealed few mutational hotspots, and most variants were associated with small perturbations in the FIX protein structure. The updated database will assist clinicians and researchers in assessing treatments for patients with hemophilia B.

SARS-CoV-2 Omicron triggers cross-reactive neutralization and Fc effector functions in previously vaccinated, but not unvaccinated, individuals.

The SARS-CoV-2 Omicron variant escapes neutralizing antibodies elicited by vaccines or infection. However, whether Omicron triggers cross-reactive humoral responses to other variants of concern (VOCs) remains unknown. We used plasma from 20 unvaccinated and 7 vaccinated individuals infected by Omicron BA.1 to test binding, Fc effector function, and neutralization against VOCs. In unvaccinated individuals, Fc effector function and binding antibodies targeted Omicron and other VOCs at comparable levels. However, Omicron BA.1-triggered neutralization was not extensively cross-reactive for VOCs (14- to 31-fold titer reduction), and we observed 4-fold decreased titers against Omicron BA.2. In contrast, vaccination followed by breakthrough Omicron infection associated with improved cross-neutralization of VOCs with titers exceeding 1:2,100. This has important implications for the vulnerability of unvaccinated Omicron-infected individuals to reinfection by circulating and emerging VOCs. Although Omicron-based immunogens might be adequate boosters, they are unlikely to be superior to existing vaccines for priming in SARS-CoV-2-naive individuals.

Ad26.COV2.S breakthrough infections induce high titers of neutralizing antibodies against Omicron and other SARS-CoV-2 variants of concern.

The Janssen (Johnson & Johnson) Ad26.COV2.S non-replicating viral vector vaccine has been widely deployed for COVID-19 vaccination programs in resource-limited settings. Here we confirm that neutralizing and binding antibody responses to Ad26.COV2.S vaccination are stable for 6 months post-vaccination, when tested against multiple SARS-CoV-2 variants. Secondly, using longitudinal samples from individuals who experienced clinically mild breakthrough infections 4 to 5 months after vaccination, we show dramatically boosted binding antibodies, Fc effector function, and neutralization. These high titer responses are of similar magnitude to humoral immune responses measured in convalescent donors who had been hospitalized with severe illness, and are cross-reactive against diverse SARS-CoV-2 variants, including the neutralization-resistant Omicron (B.1.1.529) variant that currently dominates global infections, as well as SARS-CoV-1. These data have implications for population immunity in areas where the Ad26.COV2.S vaccine has been widely deployed, but where ongoing infections continue to occur at high levels.

17 O NMR Studies of Yeast Ubiquitin in Aqueous Solution and in the Solid State.

We report a general method for amino acid-type specific 17 O-labeling of recombinant proteins in Escherichia coli. In particular, we have prepared several [1-13 C,17 O]-labeled yeast ubiquitin (Ub) samples including Ub-[1-13 C,17 O]Gly, Ub-[1-13 C,17 O]Tyr, and Ub-[1-13 C,17 O]Phe using the auxotrophic E. coli strain DL39 GlyA λDE3 (aspC- tyrB- ilvE- glyA- λDE3). We have also produced Ub-[η-17 O]Tyr, in which the phenolic group of Tyr59 is 17 O-labeled. We show for the first time that 17 O NMR signals from protein terminal residues and side chains can be readily detected in aqueous solution. We also reported solid-state 17 O NMR spectra for Ub-[1-13 C,17 O]Tyr and Ub-[1-13 C,17 O]Phe obtained at an ultrahigh magnetic field, 35.2 T (1.5 GHz for 1 H). This work represents a significant advance in the field of 17 O NMR studies of proteins.

Qualitative Research to Explore the Patient Experience of X-Linked Hypophosphatemia and Evaluate the Suitability of the BPI-SF and WOMAC® as Clinical Trial End Points.

BACKGROUND: X-linked hypophosphatemia (XLH) is a rare genetic disorder characterized by renal phosphate wasting and defective bone mineralization. Symptoms include bone pain, joint pain, stiffness, and fatigue. Published evidence regarding the patient experience of XLH is sparse and no XLH-specific outcome measures have been validated. OBJECTIVES: To understand the symptoms, impacts, and patient experience of XLH and to evaluate the face and content validity of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC®) and the Brief Pain Inventory Short Form (BPI-SF) for use as end points in XLH clinical trials. METHODS: Face-to-face, qualitative, semistructured interviews were conducted with 18 adults with XLH in the United States using concept elicitation and cognitive debriefing techniques. Open-ended questioning elicited spontaneous concepts focusing on XLH-associated symptoms and functional limitations. Cognitive debriefing of the WOMAC® and BPI-SF assessed the relevance and patient understanding of item wording, recall period, and response options. RESULTS: Various distinct symptom concepts were elicited including pain symptoms, dental symptoms, sensory symptoms, tiredness/fatigue symptoms, and musculoskeletal symptoms. Participants reported experiencing significant bone and joint pain, stiffness, mobility limitations, and an impact on their ability to work. Cognitive interviewing found both instruments to be relevant and well understood by most patients. CONCLUSIONS: The interviews generated rich, qualitative insights into the patient experience of XLH. Cognitive debriefing of the BPI-SF and WOMAC® supported their value as XLH clinical trial end points. Future research will assess the psychometric properties of these instruments for use in the XLH population.

Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision.

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core ß-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens.

Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.

Diverse specificity of cellulosome attachment to the bacterial cell surface.

During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

Structure of the small Dictyostelium discoideum myosin light chain MlcB provides insights into MyoB IQ motif recognition.

Dictyostelium discoideum MyoB is a class I myosin involved in the formation and retraction of membrane projections, cortical tension generation, membrane recycling, and phagosome maturation. The MyoB-specific, single-lobe EF-hand light chain MlcB binds the sole IQ motif of MyoB with submicromolar affinity in the absence and presence of Ca(2+). However, the structural features of this novel myosin light chain and its interaction with its cognate IQ motif remain uncharacterized. Here, we describe the NMR-derived solution structure of apoMlcB, which displays a globular four-helix bundle. Helix 1 adopts a unique orientation when compared with the apo states of the EF-hand calcium-binding proteins calmodulin, S100B, and calbindin D9k. NMR-based chemical shift perturbation mapping identified a hydrophobic MyoB IQ binding surface that involves amino acid residues in helices I and IV and the functional N-terminal Ca(2+) binding loop, a site that appears to be maintained when MlcB adopts the holo state. Complementary mutagenesis and binding studies indicated that residues Ile-701, Phe-705, and Trp-708 of the MyoB IQ motif are critical for recognition of MlcB, which together allowed the generation of a structural model of the apoMlcB-MyoB IQ complex. We conclude that the mode of IQ motif recognition by the novel single-lobe MlcB differs considerably from that of stereotypical bilobal light chains such as calmodulin.

Functional redundancy between the transcriptional activation domains of E2A is mediated by binding to the KIX domain of CBP/p300.

The E-protein transcription factors play essential roles in lymphopoiesis, with E12 and E47 (hereafter called E2A) being particularly important in B cell specification and maturation. The E2A gene is also involved in a chromosomal translocation that results in the leukemogenic oncoprotein E2A-PBX1. The two activation domains of E2A, AD1 and AD2, display redundant, independent, and cooperative functions in a cell-dependent manner. AD1 of E2A functions by binding the transcriptional co-activator CBP/p300; this interaction is required in oncogenesis and occurs between the conserved ϕ-x-x-ϕ-ϕ motif in AD1 and the KIX domain of CBP/p300. However, co-activator recruitment by AD2 has not been characterized. Here, we demonstrate that the first of two conserved ϕ-x-x-ϕ-ϕ motifs within AD2 of E2A interacts at the same binding site on KIX as AD1. Mutagenesis uncovered a correspondence between the KIX-binding affinity of AD2 and transcriptional activation. Although AD2 is dispensable for oncogenesis, experimentally increasing the affinity of AD2 for KIX uncovered a latent potential to mediate immortalization of primary hematopoietic progenitors by E2A-PBX1. Our findings suggest that redundancy between the two E2A activation domains with respect to transcriptional activation and oncogenic function is mediated by binding to the same surface of the KIX domain of CBP/p300.

Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structure.

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.

Conformational analysis of StrH, the surface-attached exo-ß-D-N-acetylglucosaminidase from Streptococcus pneumoniae.

Streptococcus pneumoniae is a serious human pathogen that presents on its surface numerous proteins involved in the host-bacterium interaction. The carbohydrate-active enzymes are particularly well represented among these surface proteins, and many of these are known virulence factors, highlighting the importance of carbohydrate processing by this pathogen. StrH is a surface-attached exo-ß-D-N-acetylglucosaminidase that cooperates with the sialidase NanA and the ß-galactosidase BgaA to sequentially degrade the nonreducing terminal arms of complex N-linked glycans. This enzyme is a large multi-modular protein that is notable for its tandem N-terminal family GH20 catalytic modules, whose individual X-ray crystal structures were recently reported. StrH also contains C-terminal tandem G5 modules, which are uncharacterized. Here, we report the NMR-determined solution structure of the first G5 module in the tandem, G5-1, which along with the X-ray crystal structures of the GH20 modules was used in conjunction with small-angle X-ray scattering to construct a pseudo-atomic model of full-length StrH. The results reveal a model in which StrH adopts an elongated conformation that may project the catalytic modules away from the surface of the bacterium to a distance of up to ~250 Å.

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1.

E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein-encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein-driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1-induced leukemia.

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance.

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm.

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine.

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.

Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: calcium-induced effects on conformation and target recognition.

The assembly of a functional cellulose-degrading complex termed the cellulosome involves two specific calcium-dependent cohesin-dockerin interactions: type I and type II. Extensive structural and mutagenesis studies have been performed on the type I modules and their interaction in an attempt to identify the underlying molecular determinants responsible for this specificity. However, very little structural information exists for the type II interaction. We have performed a variety of biophysical studies on the type II dockerin-X-module modular pair (DocX), which comprises the C-terminal region of cellulosomal scaffoldin subunit from Clostridium thermocellum, to determine the effect of calcium on its structure and interaction with type II cohesin. Our results indicate that calcium binding to type II dockerin occurs with an apparent dissociation constant (K(d)) of 7 microM, induces stable secondary and tertiary structure, and leads to the exposure of a hydrophobic surface. Calcium binding also results in the homodimerization of DocX. Analytical ultracentrifugation experiments indicate that the DocX homodimer has an elongated shape and a K(d) of approximately 40 microM. However, addition of the SdbA type II cohesin binding partner led to the dissociation of the DocX homodimer and to the formation of a 1:1 heterodimer. We propose that the exposed hydrophobic surface forms, at least in part, the type II cohesin-binding site, which in the absence of cohesin results in the dimerization of DocX.

Purification and crystallization of a trimodular complex comprising the type II cohesin-dockerin interaction from the cellulosome of Clostridium thermocellum.

The high-affinity calcium-mediated type II cohesin-dockerin interaction is responsible for the attachment of the multi-enzyme cellulose-degrading complex, termed the cellulosome, to the cell surface of the thermophilic anaerobe Clostridium thermocellum. A trimodular 40 kDa complex comprising the SdbA type II cohesin and the the CipA type II dockerin-X module modular pair from the cellulosome of C. thermocellum has been crystallized. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.21, b = 52.34, c = 154.69 A. The asymmetric unit contains one molecule of the protein complex and native and selenomethionine-derivative crystals diffracted to 2.1 and 2.0 A, respectively.