Adams et al. 10.1073/pnas.0507109103. |
Supporting Table 1
Supporting Table 2
Supporting Methods
Supporting Figure 5
Supporting Figure 6
Supporting Figure 5
Fig. 5. Affinity and thermostability of the type-II Coh–XDoc interaction. (a Upper) The isothermal calorimetric trace for the titration of XDoc into a cell containing type-II Coh at 55°C. (Lower) The binding isotherms versus molar ratio of XDoc after correction for heat of dilution of XDoc and normalization of the amount of XDoc injected (filled diamonds). The curve represents the best fit to a single-site model. The lone data point in the transition region of the curve represents the extraordinary high affinity of this interaction. (b) Differential scanning calorimetric denaturation profiles of 2.5 mM type-II Coh (rose), 1.55 mM XDoc (blue), and 5 mM type-II Coh-in the presence of 2.5 mM XDoc (green). All samples were in the presence of 5 mM CaCl2.
Fig. 6.
Sequence alignment of XDoc modular pairs. The secondary structures of the X module and type-II Doc are depicted above the alignments with arrows and rectangles representing b-strands and a-helices, respectively. Identical and similar conserved residues are shaded in yellow. The residues located at the XDoc interface are highlighted in green boxes. Residues forming aliphatic contacts at the Coh–Doc interface are defined by blue bold lettering, and residues involved in the hydrogen-bond network are highlighted in red boxes. Sequences compared are CipA–XDoc modular pair from Clostridium thermocellum CipA scaffoldin subunit (NCBI accession no. Q06851); ScaA–XDoc modular pair from Acetivibrio cellulolyticus ScaA scaffoldin subunit (NCBI accession no. AF155197); Igd2–XDoc modular pair from the C. thermocellum putative gene-product Ig-like-containing protein (NCBI accession no. ZP00504513).Table 1. Structure statistics
Refinement statistics | |
Resolution, Å | 25.0–2.1 |
Reflections | 21457 |
Space group | P2 12121 |
Unit cell (Å) | a = 45.57, b = 52.22, c = 156.26 |
Protein atoms | 2,458 |
Solvent molecules | 309 |
Solvent content, % | 47.4 |
R value, % | 20.4 |
Rfree value*, % | 24.3 |
Average B factor, Å2 | 19.9 |
Deviations from ideal geometery† | |
Bond lengths, Å | 0.006 |
Bond angles, ° | 1.3 |
Ramachandran‡ | |
Most favored regions, % | 85.7 |
Additional allowed regions, % | 13.6 |
Generously allowed regions, % | 0.7 |
Disallowed regions, % | 0.0 |
*Five percent of reflections were randomly chosen for calculation of Rfree value.
†
Root-mean-squared deviation from ideal geometry (1).‡
Percentage of residues in regions of the Ramachandran plot (2).
1. Engh, R. A. & Huber, R. (1991) Acta Crystallogr. A 47, 392–400.
2. Laskowsky, R. A., McArthur, M. W., Moss, D. S. & Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283–291.
Table 2. Coh–Doc interface hydrogen bond contacts
Cohesin | Water | Dockerin | X module |
Gln-52 Ne | Asn-122 Od | ||
Gln-52 Oe2 | Met-144 Nd | ||
Gln-52 Oe 2 | Wat-2 | Gln-145 Nd | |
Asp-97 Od2 | Asn-122 Nd | ||
Asn-106 Od | Asn-122 N | ||
Tyr-111 O | His-151 N | ||
Ala-115 N | Wat-30 | His-151 O | |
Ser-151 O | Asn-143N 2d | ||
Pro-153 O | Wat-31 | Asn-143 N 2d | |
Pro-153 O | Wat-31 | Gln-145 O 1e | |
Pro-153 O | Wat-122 | Gln-145 O 1e | |
Gln-160 Oe 1 | Wat-122 | Gln-145 O 1e | |
Asp-165 O | Lys-152 Nz | ||
Gly-166 O | Wat-5 | Gln-145 O | |
Glu-167 Oe 2 | Lys-152 Nz | ||
Glu-167 Oe 2 | Wat-14 | Ser-20 N | |
Glu-167 Oe 2 | Ser-20 Og |
Supporting Methods
Isothermal Titration and Differential Scanning Calorimetry.
The type-II cohesin (Coh) and X module–type-II dockerin (XDoc) protein samples were prepared in 25 mM Hepes, pH 7.5, 50 mM NaCl, 5 mM CaCl2, 1 mM b-mercaptoethanol that was filtered and degassed. Titration of XDoc (75 mM) into type-II Coh (5 mM) was performed at 30°C and 55°C by using a VP-ITC titration calorimeter from Microcal (Amherst, MA). 40 injections of 5 ml were made with 240 s of equilibration between injections. Integration of the thermogram, after correction for heats of dilution, yielded a binding isotherm that fit best to a one-site binding model by using the isothermal titration calorimetry (ITC) data-analysis program origin 5.0 (Microcal). A nonlinear least-squares-curve-fitting algorithm was used to determine the stoichiometric ratio, the association constant (Ka), and the change in enthalpy of the interaction. The heat-capacity measurements of 2.5 mM Coh, 1.55 mM XDoc, and 5 mM Coh in the presence of 2.5 mM XDoc were performed from 20°C to 110°C at a rate of 45°C per hour by using a VP-DSC differential scanning calorimeter from Microcal. The thermodynamic parameters of the single-folding transitions were calculated by using the program origin 5.0 software (Microcal). The binding affinity of the Coh–XDoc complex was determined by using the model described previously for a high-affinity protein–protein interaction where the melting temperature of the complex is higher than the melting temperatures of the individual components, taking into account the temperature shift of both transitions (36).