Basis for accurate protein pKa prediction with machine learning

30 January 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


pH regulates protein structures and the resulting functions in many biological processes via protonation and deprotonation of ionizable side chains where the titration equilibra is determined by pKa. To accelerate pH-dependent molecular mechanism research in life science or industrial protein and drug designs, fast and accurate pKa prediction is crucial. Here we present a theoretical pK data set PHMD549, which was successfully applied to four distinct machine learning methods, including DeepKa that was proposed in our previous work. To reach a valid comparison, EXP67S was selected as the test set. Encouragingly, DeepKa was improved significantly and outperforms other state-of-the-art methods, except for the constant-pH molecular dynamics, which was utilized to create PHMD549. More importantly, DeepKa reproduced experimental pKa orders of acidic dyads in five enzyme catalytic sites. Apart from structural proteins, DeepKa was found applicable to intrinsically disordered peptides. Further, in combination with solvent exposures, it's revealed that DeepKa offers the most accurate prediction under the challenging circumstance that hydrogen bonding or salt bridge interaction is partly compensated by desolvation for a buried side chain. Finally, our benchmark data qualify PHMD549 and EXP67S as the basis for future developments of protein pKa prediction tools driven by artificial intelligence. In addition, DeepKa built on PHMD549 has been proved an efficient protein pKa predictor and thus can be applied immediately to, for example, pKa database construction, protein design, drug discovery and so on.


molecular dynamics
deep learning
pKa prediction

Supplementary materials

Supporting Information
Supplemental figures and tables including statistics of ionizable residues in proteins, explanation of Henderson-Hasselbalch equation, pKa convergence plot by CpHMD simulations, statistics of pKa database PHMD549, overlaps of four test sets, structures and pKa's of two HEWL proteins, residue type-specific assessment of DeepKa, pKa values of acids in NUPR1 protein, RMSD estimation for PKAI+ and pKa shift distributions and solvent accessibility calculations for PDB and AlphaFold structures.

Supplementary weblinks


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