Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

Suk Kyu Ko; Carolin Berner; Alina Kulakova; Markus Schneider; Iris Antes; Gerhard Winter; Pernille Harris; Günther H J Peters

doi:10.1016/j.csbj.2022.03.012

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

Comput Struct Biotechnol J. 2022 Mar 17:20:1439-1455. doi: 10.1016/j.csbj.2022.03.012. eCollection 2022.

Authors

Suk Kyu Ko¹, Carolin Berner², Alina Kulakova³, Markus Schneider⁴, Iris Antes⁴, Gerhard Winter², Pernille Harris³, Günther H J Peters¹

Affiliations

¹ Technical University of Denmark, Department of Chemistry, 2800 Kongens Lyngby, Denmark.
² Ludwig Maximilian University of Munich, Department of Pharmacy, 81377 Munich, Germany.
³ University of Copenhagen, Department of Chemistry, 2100 Copenhagen, Denmark.
⁴ Technical University of Munich, TUM School of Life Sciences, 85354 Freising, Germany.

Abstract

Granulocyte-colony stimulating factor (GCSF) is a widely used therapeutic protein to treat neutropenia. GCSF has an increased propensity to aggregate if the pH is increased above 5.0. Although GCSF is very well experimentally characterized, the exact pH-dependent aggregation mechanism of GCSF is still under debate. This study aimed to model the complex pH-dependent aggregation behavior of GCSF using state-of-the-art simulation techniques. The conformational stability of GCSF was investigated by performing metadynamics simulations, while the protein-protein interactions were investigated using coarse-grained (CG) simulations of multiple GCSF monomers. The CG simulations were directly compared with small-angle X-ray (SAXS) data. The metadynamics simulations demonstrated that the orientations of Trp residues in GCSF are dependent on pH. The conformational change of Trp residues is due to the loss of Trp-His interactions at the physiological pH, which in turn may increase protein flexibility. The helical structure of GCSF was not affected by the pH conditions of the simulations. Our CG simulations indicate that at pH 4.0, the colloidal stability may be more important than the conformational stability of GCSF. The electrostatic potential surface and CG simulations suggested that the basic residues are mainly responsible for colloidal stability as deprotonation of these residues causes a reduction of the highly positively charged electrostatic barrier close to the aggregation-prone long loop regions.

Keywords: A3D, aggrescan3d; Aggregation; CD, circular dichroism; CG, coarse-grained; COM, center of mass; CV, collective variable; Coarse grained simulations; D0, infinite dilution diffusion coefficient; DLS, dynamic light scattering; DSF, differential scanning fluorimetry; ESRF, European Synchrotron Radiation Facility; FES, free energy surface; FF, force field; GCSF, granulocyte-colony stimulating factor; Granulocyte stimulating factor; HDX, hydrogen deuterium exchange; MD, molecular dynamics; Molecular dynamics; NMR, nuclear magnetic resonance; PDB, Protein Data Bank; PME, particle-mesh-Ewald; PPI, protein-protein interaction; SAP, spatial aggregation propensity; SAXS, small-angle X-ray scattering; SIRAH, South American Initiative for a Rapid and Accurate Hamiltonian; Small angle X-ray scattering; cMD, conventional molecular dynamics; r(H)0, infinite dilution values for the hydrodynamic radius.