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. 2022 Dec 14:9:960248.
doi: 10.3389/fmolb.2022.960248. eCollection 2022.

Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint

Alessandro T Caputo  1   2 , Roberta Ibba  1   3 , James D Le Cornu  1   4 , Benoit Darlot  1 , Mario Hensen  1 , Colette B Lipp  1 , Gabriele Marcianò  5 , Snežana Vasiljević  1 , Nicole Zitzmann  1 , Pietro Roversi  6   7
Affiliations

Crystal polymorphism in fragment-based lead discovery of ligands of the catalytic domain of UGGT, the glycoprotein folding quality control checkpoint

Alessandro T Caputo et al. Front Mol Biosci. .

Abstract

None of the current data processing pipelines for X-ray crystallography fragment-based lead discovery (FBLD) consults all the information available when deciding on the lattice and symmetry (i.e., the polymorph) of each soaked crystal. Often, X-ray crystallography FBLD pipelines either choose the polymorph based on cell volume and point-group symmetry of the X-ray diffraction data or leave polymorph attribution to manual intervention on the part of the user. Thus, when the FBLD crystals belong to more than one crystal polymorph, the discovery pipeline can be plagued by space group ambiguity, especially if the polymorphs at hand are variations of the same lattice and, therefore, difficult to tell apart from their morphology and/or their apparent crystal lattices and point groups. In the course of a fragment-based lead discovery effort aimed at finding ligands of the catalytic domain of UDP-glucose glycoprotein glucosyltransferase (UGGT), we encountered a mixture of trigonal crystals and pseudotrigonal triclinic crystals-with the two lattices closely related. In order to resolve that polymorphism ambiguity, we have written and described here a series of Unix shell scripts called CoALLA (crystal polymorph and ligand likelihood-based assignment). The CoALLA scripts are written in Unix shell and use autoPROC for data processing, CCP4-Dimple/REFMAC5 and BUSTER for refinement, and RHOFIT for ligand docking. The choice of the polymorph is effected by carrying out (in each of the known polymorphs) the tasks of diffraction data indexing, integration, scaling, and structural refinement. The most likely polymorph is then chosen as the one with the best structure refinement Rfree statistic. The CoALLA scripts further implement a likelihood-based ligand assignment strategy, starting with macromolecular refinement and automated water addition, followed by removal of the water molecules that appear to be fitting ligand density, and a final round of refinement after random perturbation of the refined macromolecular model, in order to obtain unbiased difference density maps for automated ligand placement. We illustrate the use of CoALLA to discriminate between H3 and P1 crystals used for an FBLD effort to find fragments binding to the catalytic domain of Chaetomium thermophilum UGGT.

Keywords: UGGT; [(morpholin-4yl)methyl]quinolin-8-ol; crystal polymorphism; structure determination pipeline; structure-based lead discovery.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Sequence alignment of GT24 domains of a few eukaryotic UGGTs. Ct: Chaetomium thermophilum; Hs: Homo sapiens; Mm: Mus musculus; At: Arabidopsis thaliana; Ce: Caenorhabditis elegans; Dm: Drosophila melanogaster; Sp: Schizosaccharomyces pombe; Ca: Candida albicans. The CtUGGT D1302 and D1304 residues coordinating the catalytic Ca2+ ion are completely conserved across these sequences. Red triangles mark the CtUGGT 1346WY1347 clamp. A blue square marks the position of CtUGGT Y1211 (coordinating the UDP–Glc uracyl ring). A green circle marks the position of CtUGGT D1435 (coordinating the Ca2+ ion).
FIGURE 2
FIGURE 2
(A,B) Related crystal symmetries and lattices of the H3 (cyan) and P1 (orange) crystal forms of the CtUGGT GT24 crystals. (C)The molecule in the asymmetric unit of the H3 crystal is shown, together with two of its symmetry mates (cyan), in cartoon representation. This portion of the H3 lattice has been overlaid onto the asymmetric unit of the P1 crystals (three chains, painted orange, light orange, and yellow-orange; also in cartoon representation) by superposing the “A” of the H3 crystal to the “A” chain in the P1 crystal.
FIGURE 3
FIGURE 3
CtUGGTGT24 UDP−Glc  and CtUGGTGT24 5M−8OH−Q crystal structures. (A) CtUGGTGT24 UDP−Glc (PDB ID 6FSN). Protein atoms in stick representation; C cyan (but UDP–Glc C magenta), O red, N blue, P orange. H-bonds and Ca-coordination bonds are in yellow dashed lines. At the top, the residues coordinating the uracyl ring: the side chain of CtUGGT Y1211 and the main chain of S1207. At the bottom, the Ca2+ ion is a green sphere, and its coordinating water molecules are red spheres. The side chains of residues D1302, D1304, and D1435 coordinate the Ca2+. Three more coordination sites are taken up by the β phosphate, the O2′ atom of its Glc ring, and a water molecule. The uracyl O4 atom accepts an H-bond from the S1207 main chain NH. (B) Zoom onto the CtUGGT 1346YW1347 clamp (C atoms in cyan) binding 5M-8OH-Q (C atoms in green). The 8OH-quinoline ring inserts and is sandwiched between the aromatic side chains of the conserved residues 1346YW1347. The two aromatic side chains stabilise the quinoline ring, forming an aromatic trimer; the 8-OH group of the quinoline also establishes an H-bond to the side chain of 1402H. Representative distances to interacting residues are in green dashed lines. Only two of the many morpholine ring placements are shown. The unbiased Fo–Fc map is represented as a grey mesh at a 2.0 σ contour level. PDB ID: 7ZXW.
FIGURE 4
FIGURE 4
Ligands x0441 and x0763 bind to surface pockets of the CtUGGT GT24 domain. The crystal structures of CtUGGTGT24 UDP−Glc soaked in compounds x0441 and x0763 are painted in green and cyan cartoon representation, respectively. The molecule of UDP–Glc in the catalytic pocket of each structure is represented in sticks (its C atoms also in green and cyan for the x0441 and x0763 soaked crystal structure, respectively; N atoms in blue, O atoms in red, and P atoms in orange). Two partially overlapping poses of compound x0673 are represented in sticks with magenta C atoms. Two partially overlapping poses of compound x0441 are represented in sticks with yellow C atoms, next to CtUGGT GT24 residue H1042, also represented in sticks.
FIGURE 5
FIGURE 5
(A) Box plots of resolution for 390 CtUGGT GT24 FBLD datasets, which refined better in H3, and 49 datasets, which refined better in P1. (B) Box plots of Rmeas and Rfree for the same CtUGGT GT24 FBLD datasets. Both the resolution and R scales are logarithmic.
FIGURE 6
FIGURE 6
CoALLA flow diagram.
See this image and copyright information in PMC

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