Biology subjects

Gizzio, J.

Publications and source records attributed to Gizzio, J..

3 recordsLinked to original sources

bioRxiv2026

Defining the Active Conformation of Typical Protein Kinases Domains from Substrate-Bound PDB Structures Enables Active-State AlphaFold2 Models for All 437 Human Catalytic Protein Kinases

Humans have 437 catalytically competent protein kinase domains with the typical kinase fold, similar to the structure of Protein Kinase A (PKA). The active form of a kinase must satisfy requirements for binding ATP, magnesium, and substrate. From structural bioinformatics analysis of 248 crystal structures of 54 unique substrate-bound kinases, we derived structural criteria for the active form of typical protein kinases. We include well-known requirements on the DFG motif of the activation loop and the N-terminal domain salt bridge, but also on the positions of the N-terminal and C-terminal segments of the activation loop that must be placed appropriately to bind substrate. With these criteria, only 130 of the 437 human catalytic protein kinases (30%) are in the Protein Data Bank in their active form. Because the active forms of catalytic kinases are needed for understanding substrate specificity and the effects of mutations on catalytic activity in cancer and other diseases, we used AlphaFold2 to produce models of all 437 human protein kinases in the active form. This was accomplished with templates from the PDB that resemble substrate-bound structures, shallow multiple sequence alignments of orthologs and close paralogs of the query protein, and application of the active-kinase criteria to the output models. We selected models for each kinase based on intramolecular ipSAE scores of the activation loop residues of these models, demonstrating that the highest scoring models have the lowest or close to the lowest RMSD to 29 non-redundant substrate-bound structures in the PDB. A larger benchmark of 117 active kinase structures with solved activation loops in the PDB shows that 71% of the highest scoring AlphaFold2 models had backbone RMSD < 1.0 [A] to the benchmark structures and 92% were within 2.0 [A]. Models for all 437 catalytic kinases are available at https://dunbrack.fccc.edu/kincore/activemodels. We believe they may be useful for interpreting mutations leading to constitutive catalytic activity in cancer as well as for templates for modeling substrate and inhibitor binding for molecules which bind to the active state.

bioinformatics↗

bioRxiv2024

Evolutionary sequence and structural basis for the distinct conformational landscapes of Tyr and Ser/Thr kinases

Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches - tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory "folded" conformation, due to intrinsic sequence effects. Here we investigated the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a novel thermodynamic cycle involving many (n=108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation "DFG-out Activation Loop Folded", is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs.

biophysics↗

bioRxiv2022

Evolutionary Divergence in the Conformational Landscapes of Tyrosine vs Serine/Threonine Kinases

Inactive conformations of protein kinase catalytic domains where the DFG motif has a "DFG-out" orientation and the activation loop is folded present a druggable binding pocket that is targeted by FDA-approved "type-II inhibitors" in the treatment of cancers. Tyrosine Kinases (TKs) typically show strong binding affinity with a wide spectrum of type-II inhibitors while Serine/Threonine Kinases (STKs) usually bind more weakly which we suggest here is due to differences in the folded to extended conformational equilibrium of the activation loop between TKs vs. STKs. To investigate this, we use sequence covariation analysis with a Potts Hamiltonian statistical energy model to guide absolute binding free-energy molecular dynamics simulations of 74 protein-ligand complexes. Using the calculated binding free energies together with experimental values, we estimated free-energy costs for the large-scale ([~]17-20[A]) conformational change of the activation loop by an indirect approach, circumventing the very challenging problem of simulating the conformational change directly. We also used the Potts statistical potential to thread large sequence ensembles over active and inactive kinase states. The structure-based and sequence-based analyses are consistent; together they suggest TKs evolved to have free-energy penalties for the classical "folded activation loop" DFG-out conformation relative to the active conformation that is, on average, 4-6 kcal/mol smaller than the corresponding values for STKs. Potts statistical energy analysis suggests a molecular basis for this observation, wherein the activation loops of TKs are more weakly "anchored" against the catalytic loop motif in the active conformation, and form more stable substrate-mimicking interactions in the inactive conformation. These results provide insights into the molecular basis for the divergent functional properties of TKs and STKs, and pharmacological implications for the target selectivity of type-II inhibitors.

biophysics↗