Michal Slutzki

Single-molecule dissection of the high-affinity cohesin-dockerin complex.

Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin–dockerin rupture forces (>120 pN) are among the highest reported for a receptor–ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop–helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin’s critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein–protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.

Comparing Class A GPCRs to bitter taste receptors: Structural motifs, ligand interactions and agonist-to-antagonist ratios.

G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins that play a key role in human physiology. The GPCR superfamily comprises about 800 members, classified into several classes, with rhodopsin-like Class A being the largest and most studied thus far. A huge component of the human repertoire consists of the chemosensory GPCRs, including ∼400 odorant receptors, 25 bitter taste receptors (TAS2Rs), which are thought to guard the organism from consuming poisons, and sweet and umami TAS1R heteromers, which indicate the nutritive value of food. The location of the binding site of TAS2Rs is similar to that of Class A GPCRs. However, most of the known bitter ligands are agonists, with only a few antagonists documented thus far. The agonist-to-antagonist ratios of Class A GPCRs vary, but in general are much lower than for TAS2Rs. For a set of well-studied GPCRs, a gradual change in agonists-to-antagonists ratios is observed when comparing low (10 μM)- and high (10 nM)-affinity ligand sets from ChEMBL and the DrugBank set of drugs. This shift reflects pharmaceutical bias toward the therapeutically desirable pharmacology for each of these GPCRs, while the 10 μM sets possibly represent the native tendency of the receptors toward either agonists or antagonists. Analyzing ligand-GPCR interactions in 56 X-ray structures representative of currently available structural data, we find that the N-terminus, TM1 and TM2 are more involved in binding of antagonists than of agonists. On the other hand, ECL2 tends to be more involved in binding of agonists. This is of interest, since TAS2Rs harbor variations on the typical Class A sequence motifs, including the absence of the ECL2-TM3 disulfide bridge. This suggests an alternative mode of regulation of conformational states for TAS2Rs, with potentially less stabilized inactive state. The comparison of TAS2Rs and Class A GPCRs structural features and the pharmacology of the their ligands highlights the intricacies of GPCR architecture and provides a framework for rational design of new ligands.

Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability

The cellulosome is a large extracellular multi‐enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme‐integrating ScaA scaffoldin into the cohesin‐bearing ScaB adaptor scaffoldin. In the absence of a high‐resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N‐terminal Trp and a C‐terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca2+‐bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well‐defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.

Structural and functional characterization of a novel type‐III dockerin from Ruminococcus flavefaciens

ScaB cohesin binds to ScaADoc by enzyme linked immunosorbent assay (View interaction)

Complexity of the Ruminococcus flavefaciens FD-1 cellulosome reflects an expansion of family-related protein-protein interactions

Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.

Indirect ELISA-based approach for comparative measurement of high-affinity cohesin-dockerin interactions.

The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non‐catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell‐wall degrading complex. This interaction is species‐specific, for example, the enzyme‐borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein–protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme‐linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high‐affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (Kd) of mutants relative to wild type, a pre‐equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin–dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein–protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high‐affinity interactions. Copyright

Crucial Roles of Single Residues in Binding Affinity, Specificity, and Promiscuity in the Cellulosomal Cohesin-Dockerin Interface

Background: Cellulosomal cohesin-dockerin interactions show intraspecies promiscuity but interspecies specificity. Results: A combination of computations and experiments reveals single cohesin residue mutations with dramatic effects not only on binding affinity but also on specificity and promiscuity. Conclusion: Natural interspecies specificity barriers in the cohesin-dockerin interaction are easily overcome by single mutations, indicating considerable plasticity. Significance: This study sheds light on the malleability and evolvability of a high affinity interaction. Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn37 show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.

Standalone cohesin as a molecular shuttle in cellulosome assembly

The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin‐bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin‐borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.

Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.

The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.

Insights into a type III cohesin–dockerin recognition interface from the cellulose‐degrading bacterium Ruminococcus flavefaciens

Cellulosomes are large multicomponent cellulose‐degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self‐assembly of cellulosomes is mediated by the ultra‐high‐affinity cohesin–dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well‐studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self‐assembly in this bacterium is the dockerin ScaADoc, found on the non‐catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose‐degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C‐terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens. Copyright