Computational protocol: Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

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Protocol publication

[…] Engineered Dom-II was crystallized using the hanging-drop vapor-diffusion method and equilibrating a 1–4 µl mixture containing protein solution (11.9 mg/ml Dom-II in 20 mM Tris-HCl, pH 7.5) and reservoir solution (1.5 M Li2SO4, 0.1 M Na-HEPES salt, pH 7.5) at ratios 1∶2.5 or 1∶3 against 700 µl of reservoir solution. Crystallization of the SM5-1 Fab fragment was achieved with the sitting-drop vapor-diffusion set-up. 200 nl of protein solution (9.7 mg/ml in 20 mM Tris-HCl, pH 7.5) were mixed with 200 nl of reservoir solution (4 M sodium formate) and equilibrated against reservoir solution. Crystals of individual Dom-II and SM5-1 Fab grew at 19°C within two days. In both cases, crystals were flash-cooled in liquid nitrogen without addition of cryoprotectants.Dom-II of gB in complex with SM5-1 was crystallized with the sitting-drop vapor-diffusion method using protein concentrations between 10 and 15 mg/ml in 20 mM Tris-HCl, pH 7.5 and mixing 200 nl of protein solution with 200 nl of reservoir solutions. To increase the chances for obtaining well-diffracting crystals of the complex, we crystallized SM5-1 in complex with several different mutants of Dom-II that bind SM5-1 with similar affinities than wild-type Dom-II . However, most complexes only produced crystals that were intergrown and unsuitable for X-ray diffraction experiments. In case of the complex formed between double mutant gB Dom-II-I397A/N398A and SM5-1 Fab, single crystals could be isolated after equilibrating the protein-reservoir mixture against 70 µl reservoir solution (0.1 M HEPES, pH 7.5, 0.2 M L-proline, and 24% PEG 1500) for one month at 19°C. Crystals of the complex were briefly soaked with 20% ethylene glycol before being transferred into liquid nitrogen. In the crystal structure, and as expected, the residues at positions 397 and 398 in Dom-II do not participate in the interaction with SM5-1 Fab ().All diffraction data sets were collected using 0.5 to 0.6° rotation frames at PX beamline BL 14.1 at Hemholtz Zentrum Berlin BESSY synchrotron facility and processed with program XDS . All three crystal structures could be solved by molecular replacement using program PHASER within the CCP4 program suite , . The structure of Dom-II comprising residues 118 to 132 and 344 to 438 of the HCMV gB protein could be solved with a mixed search model that was based on the corresponding Dom-II from HSV-1 glycoprotein B (residues 142 to 152 and 364 to 459, PDB ID 2gum). The mixed model was generated using the Joint Center of Structural Genomics SCRWL server , .The unrelated Fab fragment of the human anti-HIV-1 gp120 reactive antibody E51 (PDB ID 1rzf) was used as a search model for the SM5-1 Fab structure. Flexible parts, such as CDR loops were deleted and both variable domains and constant domains were considered separately during the molecular replacement calculations. Ensemble 1: residues 109 to 210 (CL) and 114 to 214 (CH1); ensemble 2: residues 2–23, 34–54, 63–91, 96–106 (VL) and 2–23, 33–51, 57–95, 103–111 (VH). The gB Dom-II-SM5-1 Fab complex structure was solved using the crystal structures of the previously solved individual proteins as search models. Here also, any flexible regions in SM5-1 were removed and two different ensembles consisting of either the variable or constant domains used during the molecular replacement search of the SM5-1 fragment. All structures were refined with the CCP4 program REFMAC5 until convergence and until no further details could be interpreted in the electron density maps , . During the final refinement rounds a TLS refinement step was performed in order to improve the fit between the models and the experimental data.The elbow angle of the SM5-1 Fab fragment was calculated with the AS2TS web server using the following immunoglobulin domain boundaries: VL: residues 1 to 110; VH: 1 to 131, CL: 111 to 212 and CH1: 132 to 232 , . Changes in the accessible surface areas were calculated with program AREAIMOL from the CCP4 program suite using the default solvent probe radius of 1.4 Å . All molecular illustrations were generated with program PyMol ( Potential N-linked glycosylation sites were predicted using the NetNGlyc server . [...] Molecular dynamics (MD) simulations were performed for SM5-1 and for a 6-fold in silico variant (SM5-1*), in which the respective residues were exchanged to match the sequence of the less mature SM1-6 (K30T, D31G, H32Y, H115Y, N116D and R117V). For both SM5-1 and SM5-1* two independent simulations were performed starting either from the conformation present in complex with Dom-II or from the crystal structure of unbound SM5-1. This resulted in a total of four simulations. To reduce computational cost, only the VH-VL domain (aa 1–131 and 1–110 of chain H and L, respectively) of the Fab-fragment were included in the simulation. The protonation state of titratable amino acids was calculated by PROPKA and the Swiss PDB Viewer was applied to add unresolved residues and side chains, as well as for the amino acid exchanges .All MD simulations were performed using the AMBER 11 program package and the ff99SB force field parameters , . Proteins were placed in periodic truncated octahedral water boxes with at least 15 Å of solvent between any atom of the solute and the periodic box edges. The systems were neutralized by adding the respective number of Cl− ions. Initially, all four systems were minimized in a three-step procedure and then gradually heated to 310 K following a previously established protocol , . Subsequently, 100 ns MD simulations were performed with a time step of 2 fs and periodic boundaries. The Settle-algorithm was applied to constrain covalent bonds involving hydrogens. The obtained data of the simulation was analyzed and visualized using AMBER Tools and VMD . […]

Pipeline specifications

Software tools XDS, CCP4, REFMAC5, AS2TS, PyMOL, NetNGlyc, PROPKA, Swiss-PdbViewer, VMD
Applications Small-angle scattering, Protein structure analysis
Organisms Human betaherpesvirus 5, Homo sapiens