Computational protocol: Carnitine palmitoyl transferase-1A (CPT1A): a new tumor specific target in human breast cancer

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Protocol publication

[…] Homology molecular models were generated for Carnitine Palmitoyltransferase CPT1A, isoform 1 and 2 (CPT1Av1 and CPT1Av2) with two different approaches, by using ModWeb [] and SwissModel []. In the case of ModWeb, three different models have been obtained for CPT-Is1, using as templates the structures with PDB code 1ndb, 2deb and 2fy2, and two models for CPT-Is2, using 1ndb and 1t1u as templates. In the case of SwissModel the project mode approach has been used, combining the SWISS-Model pipeline tools and the program Deep-View [] as described in []. For both isoforms, the used templates have been the structures with PDB codes 1t7z (reference template), 1 nm8, 1t1u, 1xml and 2h4t. Due to the good sequence similarity between the modeled sequences and the used templates, the obtained models result similar to each other and, generally, they show good evaluation parameters (like the E-values). To individuate the final models for docking simulations, we have performed a deep inspection of the C-terms regions, where the differences between the two sequences are located, excluding all the models missing this region. Between the different models, we have selected, for the two isoforms, those obtained by using the ModWeb approach, with the protein Carnitine Acetyltransferase (PDB code 1ndb) as template. The domain at the N-terminus was missed in all the obtained models. This region has the same sequence in the two isoforms and it is predicted to constitute the membrane domain. For this reason its absence represent a slight limitation in the docking simulation. The obtained structure for CPT1-Is1 defines the coordinates for the residues between 166 and 772, that correspond to the region 30-625 in 1ndb. The sequence identity between CPT-Is1 and template is equal to 31% (identity greater than 30% are considered safety), the Model score, as obtained in Modweb, has been 1.00 (value greater than 0.7 are deemed good) and the E-value has been lower than 0.0001. Analogously, for CPT-Is2, we have obtained a model for the region from the position 166 to 755. In this case, the sequence identity has been equal to 30%, the Model score has been equal to 1.00 and the E-value has been lower than 0.0001. As model for HDAC-class I protein the structure of the HDAC-like protein (PDB Code 1c3r) has been used, without refinement. In silico docking experiments were performed using PatchDock [] and then further refined and re-ranked with FireDock []. Structural analyses and molecular graphics were performed using the program MOLMOL [].Molecular dynamics (MD) simulations of the complexes between both the CPT1A isoform (Is-1 and Is-2) and HDAC, as obtained by docking simulations, were performed using GROMACS 4.0, with the GROMOS 43a1 force field []. The simulations were performed according to previously described procedures [], except for some details. In particular, after energy minimization in vacuo, the protein was centered in a rhombic dodecahedron box (roughly 13.2 nm wide), hydrated with roughly 49000 water molecules and an opportune amount of Na+ and Cl− to assure electroneutrality and a salt concentration of about 0.01M. During simulations, the system was kept at constant temperature (300 K) and pressure (1 bar) by the Berendsen weak-coupling method []. Electrostatic interactions were calculated by the use of the particle mesh Ewald method []. The protonation state for the histidine aminoacids were obtained by means of the PDB2PQR server [], by using the PropKa algorithm. Apart the equilibration, the MD simulations were 20ns long. RMSD were calculated according to standard definitions. […]

Pipeline specifications

Software tools ModWeb, PatchDock, MOLMOL, GROMACS, PDB2PQR, PROPKA
Application Protein structure analysis
Chemicals Carnitine, Fatty Acids