Computational protocol: The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function

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Protocol publication

[…] The following concentrations and buffers were used for each sample: HD-PTPCC, 2.8 mg/ml in 20 mM HEPES pH 7.4, 300 mM NaCl, 2 mM EDTA and 2 mM DTT; HD-PTPBro1-CC, 12 mg/ml in 50 mM Tris-Cl pH 8.0, 100 mM NaCl, 2 mM EDTA and 2 mM DTT; HD-PTPBro1-CC-PRR, 7.5 mg/ml in 50 mM Tris-Cl pH 8.0, 250 mM NaCl, 2 mM EDTA and 2 mM DTT. SAXS data was collected at beamlines X33 Hamburg DESY (HD-PTPBro1-CC) and BM29 ESRF (HD-PTPCC and HD-PTPBro1-CC-PRR). Data was collected at different concentrations for each sample. We did not observe any aggregation or concentration-dependent effects as shown by the consistency in the calculated R g values at each concentration (Suppl. Table ). Data processing was performed with the ATSAS suite and ScÅtter (http://www.bioisis.net/tutorial/9). The forward scattering I(0) and the radius of gyration R g were estimated with PRIMUS using the Guinier approximation (Suppl. Fig. ). GNOM was used to compute the pairwise intra-particle distance distribution function p(r) and the maximum distance D max (Suppl. Fig. ). Particle shapes were restored ab initio using DAMMIN and GASBOR. Twenty simulations were performed and the outputs were averaged and filtered using DAMAVER to produce the final envelopes (Fig. ) with a normal spatial discrepancy value of 0.61–0.67 for the DAMMIN models and 1.4–1.8 for the GASBOR models. [...] Libraries of molecular models were generated for each construct as follows. The initial model for HD-PTPCC (residues 362–704) was built from the coordinates of its crystal structure (PDB ID 5LM2, chain B). Disordered residues from the loop connecting the H1 and H2 helices were rebuilt with standard geometry in CNS. HD-PTPCC conformational variability was explored by varying the distance between the centers of masses of two separate subdomains, CC1 encompassing the “blade” and CC2 encompassing the “shaft” (according to the nomenclature used in ref. ). A library of conformers was thus generated using torsion angle molecular dynamics (TAMD) as implemented in CNS, . For each conformer the fit to the SAXS data was calculated using FoXS, and its hydrodynamic parameters calculated with SOMO.The initial model for HD-PTPBro1-CC (residues 1–714) was built from the coordinates of the crystal structure of HD-PTPBro1 (PDB ID 3RAU) and those of the individual HD-PTPCC model with the best fit to the SAXS data (Fig. ). Residues connecting the Bro1 and CC domains were rebuilt with standard stereochemistry in CNS. An N-terminal His6 tag with the correct sequence and length and was also added to the model. A library of conformers was generated using TAMD to explore different orientations between the Bro1 and CC domains. Different lengths for the linker connecting the Bro1 and CC domains were tried to ensure effective conformational sampling between the two domains. Best results were obtained with a linker spanning residues 360 to 370. Hydrodynamic and dimensional parameters for all the HD-PTPBro1-CC models were calculated with SOMO and their sedimentation coefficients used to select suitable conformers compatible with the experimentally determined values. The selected pool of models was contrasted to the experimental SAXS profile using FoXS, as previously described.Models for HD-PTPBro1-CC-PRR were generated by adding the proximal region of PRR (up to residue 738), to the coordinates of the best HD-PTPBro1-CC individual model (Fig. ). The additional residues were modeled with standard stereochemistry in CNS and their flexible conformation was sampled with TAMD. […]

Pipeline specifications

Software tools ATSAS, DAMMIN, GASBOR, CNS, FoXS
Applications Small-angle scattering, Protein structure analysis
Diseases Huntington Disease, Neoplasms