2.2. Molecular Dynamics Simulations

A molecular dynamics simulation was performed to assess the solvent accessibility of possible C4b opsonization sites within the penton base RGD loop. A simulation for the penton base pentamer with Rosetta-based RGD loop models was performed with NAMD v2.12 on the Case Western Reserve University (CWRU) high-performance computing (HPC) cluster [40]. The molecular system was minimized for 50 ps, followed by slow heating to 300 K. A molecular dynamics simulation was run for 5 ns using the Chemistry at Harvard Molecular Mechanics (CHARMM) force field [41], with Generalized Born implicit solvent (GBIS). The solvent accessibility of the atoms in the hydroxyl groups of serines and threonines, and the amino groups of lysines and arginines, within the five RGD loops was assessed for the starting and ending coordinates with the UCSF ChimeraX v1.1 “measure sasa” command and a probe radius of 1.4 Å [37].

Molecular dynamics simulations were performed to assess the possibility that C4b would interact with multiple RGD loops on one penton base pentamer. The coordinates used for C4b were from the crystal structure (PDB: 4XAM) [39]. Six different starting models for C4b relative to a penton base pentamer were prepared for molecular dynamics simulations. Four of the starting models (models 1, 2, 4, and 5) were generated with a covalent bond between Cys1010 of C4b and a residue within one penton base RGD loop (Thr343, chain C; Arg347, chain C; Thr346, chain C; or Lys297, chain E, respectively). We used UCSF Chimera v1.13 to prepare the chosen RGD loop residue, position the sulfur atom of the reactive thioester on C4b near the appropriate atom of the RGD loop residue, form a covalent bond with the “bond sel” command, and change the chain IDs to be the same for the two covalently linked polypeptides [38]. Two starting models (models 3 and 6) were generated without a covalent bond between C4b and the penton base. For these models, the Cys1010 of C4b was positioned ~10 Å from one RGD loop of the penton base (chain C or chain E, respectively). Molecular dynamics simulations were performed with NAMD v2.12 on the Case Western Reserve University (CWRU) high-performance computing (HPC) cluster [40]. The molecular systems were minimized for 50 ps followed by slow heating to 300 K. Molecular dynamics simulations were run for 12 ns using the Chemistry at Harvard Molecular Mechanics (CHARMM) force field [41] with generalized born implicit solvent (GBIS).