Potential aggregation hot spots in recombinant human keratinocyte growth factor: a computational study

Abstract

The recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The high aggregation liability of rhKGF is manifested by loss of the monomeric state, and accumulation of the aggregated species even at moderate temperatures. Here, we analyzed the rhKGF for its vulnerability toward aggregation by detection of aggregation-prone regions (APRs) using several sequence-based computational tools including TANGO, ZipperDB, AGGRESCAN, Zyggregator, Camsol, PASTA, SALSA, WALTZ, SODA, Amylpred, AMYPDB, and structure-based tools including SolubiS, CamSol structurally corrected, Aggrescan3D and spatial aggregation propensity (SAP) algorithm. The sequence-based prediction of APRs in rhKGF indicated that they are mainly located at positions 10–30, 40–60, 61–66, 88–120, and 130–140. Mapping on the rhKGF structure revealed that most of these residues including F16-R25, I43, E45, R47-I56, F61, Y62, N66, L88-E91, E108-F110, A112, N114, T131, and H133-T140 are surface-exposed in the native state which can promote aggregation without major unfolding event, or the conformational change may occur in the oligomers. The other regions are buried in the native state and their contribution to non-native aggregation is mediated by a preceding unfolding event. The structure-based prediction of APRs using the SAP tool limited the number of identified APRs to the dynamically-exposed hydrophobic residues including V12, A50, V51, L88, I89, L90, I118, L135, and I139 mediating the native-state aggregation. Our analysis of APRs in rhKGF identified the regions determining the intrinsic aggregation propensity of the rhKGF which are the candidate positions for engineering the rhKGF to reduce its aggregation tendency.

Communicated by Ramaswamy H. Sarma

Keywords:

• In silico
• aggregation-prone regions
• aggregation hot spots
• sequence-based aggregation prediction
• structure-based aggregation prediction
• spatial aggregation propensity (SAP)
• aggregation
• unfolding
• recombinant human keratinocyte growth factor

Acknowledgments

We are deeply grateful to Pasteur Institute of Iran due to their support. We are also deeply thankful to Dr. Naresh Chennamsetty due to his technical guidance for SAP calculation.

Disclosure statement

The authors declare that they have no competing interests.

Authors’ contributions

Design of study was performed by MShD, and HR. The computational studies and data analysis was performed by MShD under the supervision of HR, and MG. Manuscript writing was mainly performed by MShD and it was revised by MG, HR, and MASh.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Additional information

Funding

This research was financially supported by a grant from Pasteur Institute of Iran (for the PhD thesis of Mansoureh Shahbazi Dastjerdeh, grant number BP-9368). The molecular dynamics simulations and data analysis were performed by the computer servers provided by the Pasteur Institute of Iran.