Analysis of protein residue conservation of C. thermocellum family 50 CBMs (Figure 5.10A) revealed that the interacting residues identified in CtCBM50 (CtCBM50Cthe_0300)were poorly
conserved except for Gly7 and Asp8. Trp11 and Tyr38 are only present in 7 and 9 of the 15
CtCBM50s, respectively. Asn35 only occurs in 4 CBMs, while being replaced by other polar or
charged amino acids that could still be involved in hydrogen bonding. Ile37, although only found in 5 CBMs, is substituted by a Leu in the remaining proteins, retaining the hydrophobic effect at this position. These observations are not completely unexpected, as the consensus sequence of LysM domains shows that, while the motif is well conserved over the first 16 amino acid residues and slightly less over the last 10, the central region is poorly conserved except for an Asn residue214.
Figure 5.10. Alignment of CBM50 family members. Primary sequence alignment of (A) C. thermocellum
family 50 CBMs and (B) CtCBM50Cthe_0300 (CtCBM50) with LysMs from other microorganisms: Bacillus
subtilis (BsSafA)177, Thermus thermophilus (TthP60)224, Enterococcus faecalis (EfAtlA)215, Cladosporium
fulvum (CfECP6)227, Pteris ryukyuensis (PrLysM2)225 and Volvox carteri (VcLysM2)228. Identity to
CtCBM50Cthe_0300 is indicated with red and yellow boxes. Residue numbers refer to the corresponding CBM sequence. CtCBM50Cthe_0300 secondary structure prediction is presented above. Red triangles identify
CtCBM50Cthe_0300 residues involved in the interaction with GlcNAc3 ligand. The sequence alignment was generated with Clustal Omega196 and rendered using Espript server229.
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When in comparison with LysMs from other microorganisms (Figure 5.10B), the same trend is observed, with only CtCBM50 interacting residues Gly7 and Asp8 being conserved. By superimposing CtCBM50-GlcNAc3 (chain B) structure with TthP60_2LysM bound to a GlcNAc6,
the interacting residues identified for TthP60 LysM1 are found at the same positions as those of
CtCBM50 (Figure 5.11). With a primary sequence identity of 37%, the only identified interacting
residues conserved are Thr9 and Gly7. However, the remaining residues that interact with the ligands seem to establish the same type of contacts, with most of the hydrogen bonding also strongly established with the protein’s main chain at the same positions, and with the aromatic ring of a Tyr, which corresponds to Trp11 in the CtCBM50 structure.
Figure 5.11. Superposition of CtCBM50 with Thermus thermophilus LysM1. Chain B of CtCBM50
bound to GlcNAc3 was superposed with LysM1 of TthP60_2LysM bound to a GlcNAc6 (PDB ID 4UZ3)224.
CtCBM50 is represented as cartoon in yellow and TthP60_2LysM in green. GlcNAc3 and GlcNAc6 are shown as stick models in light grey and dark grey, respectively, and by atom type. Residues of each protein involved in the interactions with its ligand are represented by sticks and coloured by atom type. Alignment was performed using MatchMaker tool from UCF Chimera40, with an rmsd value of 0.786.
Similar with what was observed for TthP60_2LysM, CtCBM50 seems to adopt an interchain assembly behaviour where multiple CBM modules bind to the same GlcNAc oligosaccharide. The results reported here, point to an interchain multivalent assembly induced by longer GlcNAc sequences, where the individual CBM modules bind in a cooperative manner to long ligand chains, but not to short oligosaccharides (Figure 5.12). We hypothesize that individual CtCBM50 molecules bind to longer DP GlcNAc oligosaccharides by rearranging their position, so that each module binds an optimal binding epitope that contains at least two interacting N-acetyl groups. Based on the MD calculations with PG oligosaccharides, a similar behaviour could be predicted upon the binding of CtCBM50 to PG sequences, where the CBM chains dislocate to better accommodate the ligand (Figure S5.3).
The modular protein containing CtCBM50 (Figure 5.1) shares 48% of sequence identity with
B. subtilis SafA, a LysM-containing spore coat assembly protein involved in the formation of the
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Figure 5.12. Schematic representation illustrating the hypothesized cooperative binding by CtCBM50 to (A) short and (B) long GlcNAc oligosaccharides. Experimental data points to an interchain CBM
multivalent assembly induced by longer GlcNAc sequences, where individual CBM molecules bind in a cooperative manner to long ligand chains. CtCBM50 modules would bind to longer DP GlcNAc oligosaccharides by rearranging their position, so that each module binds an optimal binding epitope that contains at least two interacting N-acetyl groups. Black triangles represent the alternating N-acetyl groups.
spores, conferring for instance its elevated resistance to heat and other unfavourable growth conditions230, this can point to a possible role of family 50 CBMs in this bacterium, as well as a
reasoning for expressing such high number of these CBMs.
5.3 Conclusions
With the present work the carbohydrate specificity of C. thermocellum family 50 CBMs was assigned to β1,4-linked GlcNAc sequences, revealing a chain-length dependency with a trisaccharide as a minimum epitope for recognition. Additionally, the first structure of a CtCBM50 was solved and in complex with a GlcNAc trisaccharide, revealing an intermolecular interaction of two CBM molecules with the GlcNAc ligand. Besides binding to chitin and chitin-derived oligosaccharide sequences, peptidoglycan binding was also attested for CtCBM50, although with less affinity. The present results suggest that CtCBM50, acting in a multimodular way, is able to form a ligand binding site comprised of up to 6 binding subsites. Key residues were identified to mediate both chitin and peptidoglycan oligosaccharide recognition by CtCBM50, with Gly7, Asp8, Asn35 and Ie37 residues providing important hydrogen bonding network mediated by main chain atoms; aromatic residues Trp11 and Tyr38 contributing to binding by stacking interactions; and relevant dispersive contacts mediated by Val4, Ile21, Ile25, Pro34 and Pro39 residues. Given the identified residues responsible for CtCBM50 ligand recognition are poorly conserved among LysM domains, our observations point out to a coherent yet adaptable recognition mechanism, dictated by the protein’s structural motifs through a critical hydrogen bonding network which results from interactions with main chain atoms and provide a contact surface with the ligand monomers. Furthermore, our results also suggest that ligand binding is favored by the multivalent assembly of CtCBM50 modules, supporting the notion of LysM domains cooperative binding.
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The integrative information derived from this work will allow to understand mechanisms of carbohydrate recognition to chitin and peptidoglycan by other members of family 50 CtCBMs, contributing to elucidating their role in C. thermocellum. Moreover, the characterization of the carbohydrate recognition by these LysMs, opens the possibility of their potential biotechnological applications.
5.4 Experimental procedure
5.4.1 Gene cloning, mutagenesis and protein purification
Family 50 CBMs were cloned, expressed and purified using the same procedure as described in section 3.4.2 of Chapter 3. For the structural studies, CtCBM50Cthe_0300 was cloned in a pET28a
plasmid (Novagen), in which the recombinant protein was generated containing a C-terminal hexa-histidine tag (His-tag). Site-directed mutants were generated using the NZYMutagenesis kit (NZYTech Ltd) according to the manufacturer’s instructions using pET28a as template. Primers used to generate the mutant DNA sequences are listed in Table S5.10. Recombinant sequences of all mutant plasmid derivatives were confirmed by sequencing to ensure that only the appropriate mutations were incorporated.
E. coli BL21 harbouring the CtCBM50Cthe_0300 encoding gene was cultured in LB containing
50 µg/mL kanamycin at 37 °C until mid-exponential phase (OD600nm = 0.6), at which point IPTG
was added to a final concentration of 1 mM. Cultures were then further incubated for 5h at 37 °C, at 150 rpm in a Gallenkamp Orbital Shaker. Cells were collected by centrifugation at 5000×g for 15 minutes at 4 ºC and the cell pellet resuspended in a 50 mM sodium HEPES buffer, pH 7.5, containing 1 M NaCl, 2 mM CaCl2 and 10 mM imidazole. CtCBM50Cthe_0300 was purified from the
cleared cell-lysate by Ni2+-immobilized IMAC. The eluted protein fractions were subjected to
SDS-PAGE on 13% (w/v) acrylamide gels, stained with Coomassie Brilliant Blue, in order to assess the purity of recombinant proteins. The fractions containing pure protein were pooled and buffer-exchanged into 50 mM MOPS buffer, pH 6, containing 50 mM NaCl and 2 mM CaCl2, for
protein stability. Amicon 3-kDa molecular-mass centrifugal membranes were used to achieve higher protein concentration.
All proteins were >95% pure as judged by SDS-PAGE and their concentrations determined from
their calculated molar extinction coefficient using the Protparam tool
(http://www.expasy.org/tools/protparam.html) at 280 nm using a SpectraDrop Micro-Volume Microplate (Molecular Devices, USA).
5.4.2 Sources of carbohydrates
Information on the GlcNAc oligosaccharides and sources included in the NGL-microarrays are given in Table S2.1. Insoluble chitin polysaccharide from shrimp shell was purchased from
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Sigma-Aldrich. Peptidoglycan samples from E. coli231 and S. aureus232 were kindly provided by
Professor Sérgio Filipe (UCIBIO, NOVA).
5.4.3 Carbohydrate microarray analysis
The NGL-microarrays results exhibited correspond to the experiments presented in Chapter 3, performed as described in section 3.2.4. The results reported here, correspond to at least two independent experiments, performed with different batches of CBMs.
5.4.4 Crystallization and X-ray Diffraction Data Collection
CtCBM50Cthe_0300 complex with GlcNAc was produced by overnight incubation of the protein
(3.5 mg/mL) with β1,4-linked GlcNAc trisaccharide (GlcNAc3) at 1:2 molar ratio. Crystallization
assays were performed using an automated nano-drop dispenser Oryx8 (Douglas Instruments) and commercial screenings JBScreen Classic 2-5 (Jena Bioscience) and Structure 1 & 2 (Molecular Dimensions). 192 conditions with and without ligand were tested using the sitting-drop vapor diffusion method (SWISSCI 'MRC' 2-Drop Crystallization Plates – 96 wells, Douglas Instruments), in a 2 μL drop (containing 50% protein). Crystals of the CtCBM50-GlcNAc3 complex
grew through the course of three weeks, at 20 °C, in a crystallization condition composed of 0.1 M sodium acetate buffer, pH 4.6, and 2 M ammonium sulphate. Crystals were harvested using a solution of 0.1 M sodium acetate buffer, pH 4.6, and 2.5 M ammonium sulphate, and then flash-cooled in liquid nitrogen using 30% (v/v) glycerol as cryoprotectant added to the harvesting solution.
X-ray diffraction data from a single crystal of the CtCBM50-GlcNAc3 complex was collected under
a nitrogen stream at 100 K in ID29 beamline at the ESRF (Grenoble, France) to a maximum resolution of 1.45 Å and using X-ray radiation at a fixed wavelength of 0.9677 Å. The
CtCBM50-GlcNAc3 crystal indexed in space group C2, with cell constants a = 99.39, b = 41.77,
and c = 42.87 Å and β = 96.89°, corresponding to a calculated Matthews coefficient of 2.28 Å3/Da
and a solvent content of 46%. Data collection, processing, model building and validation statistics are shown in Table 5.1.