Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase

Abstract

Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that bind specific types of carbohydrates. Ultra high-resolution X-ray crystallographic studies of CBMs have helped to decipher the basis for specificity in carbohydrate–protein interactions. However, additional studies are needed to better understand which structural determinants confer which carbohydrate-binding properties. To address these issues, neutron crystallographic studies were initiated on one experimentally engineered CBM derived from a xylanase, X-2 L110F, a protein that is able to bind several different plant carbohydrates such as xylan, β-glucan and xyloglucan. This protein evolved from a CBM present in xylanase Xyn10A ofRhodothermus marinus. The protein was complexed with a branched xyloglucan heptasaccharide. Large single crystals of hydrogenous protein (∼1.6 mm3) were grown at room temperature and subjected to H/D exchange. Both neutron and X-ray diffraction data sets were collected to 1.6 Å resolution. Joint neutron and X-ray refinement usingphenix.refineshowed significant density for residues involved in carbohydrate binding and revealed the details of a hydrogen-bonded water network around the binding site. This is the first report of a neutron structure of a CBM and will add to the understanding of protein–carbohydrate binding interactions.

From Initial Hit to Crystal Optimization with Microseeding of Human Carbonic Anhydrase IX—A Case Study for Neutron Protein Crystallography

“…A drop of protein is mixed with precipitant and equilibrated against the reservoir in a sealed environment. In the sitting drop format it can be scaled up to almost any volume and has been reported to be successful in 100-1000 µL drops [5][6][7]. Batch crystallization has also been used, and here the protein is mixed with precipitant and optionally covered with a layer of oil.…”

“…Crystals 2018, 8, x FOR PEER REVIEW 2 of 12 [5][6][7]. Batch crystallization has also been used, and here the protein is mixed with precipitant and optionally covered with a layer of oil.…”

Neutron Crystallographic Studies Reveal Hydrogen Bond and Water-Mediated Interactions between a Carbohydrate-Binding Module and Its Bound Carbohydrate Ligand

“…13 The structure of this complex has in the past been determined to ultrahigh resolution (1.0 Å) using X-ray crystallography, 13 but even at this resolution, important Hbonding interactions had to be inferred as the H themselves were not readily visible. 10 We now report on the first neutron structure of a CBM, determined to 1.6 Å resolution, and discuss the details of ligand binding interactions. Table 1 contains a summary of all interactions between XXXG and X-2 Leu110Phe.…”

“…Despite the benefits of using neutron crystallography in structural biology, there are challenges remaining related to the preparation of large crystals (∼1 mm 3 ) for neutron studies. We were recently able to prepare large (∼1.6 mm 3 ) H/D-exchanged crystals of a genetically engineered type B CBM (X-2 with a Leu to Phe mutation at position 110, X-2 Leu110Phe) derived from CBM4-2 of the Xyn10 xylanase from Rhodothermus marinus in complex with a xyloglucan heptasaccharide ligand [XXXG shown in Figures S1 and S2; α- d -Xylp-(1–6)-β- d -Glcp-(1–4)­[α- d -Xylp-(1–6)]-β- d -Glcp-(1–4)­[α- d -Xylp-(1–6)]-β- d -Glcp-(1–4)-β- d -Glcp]. , This module belongs to the family 4 CBM as defined by the Carbohydrate-Active enZYmes Database (CAZY; ). CBM X-2 Leu110Phe, like CBM4-2, is a module with affinity for a range of carbohydrates (features of these modules are summarized in Table S1).…”