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3D structure analysis of PAKs: A clue to the rational design for affinity reagents and blockers.

ABSTRACT: The p21-activated kinase (PAK) family plays a versatile role in cell signaling by forming a hub of interactions. PAKs bind the GTPases like RAC and CDC42. Their proline-rich motifs bind SH3 adaptor proteins such as PIX and NCK. PAKs display nuclear localization signal sites and a potential Integrin binding site. No fully complete structure of the PAKs has been published; partial 3D structures of the PAK family kinases include portions of the auto-inhibited PAK1, GTPase bound to small peptides from PAKs, and the kinase domains from PAK1 and PAK4-6 (with small ligands in a few cases). This review focuses on exploring the intermolecular interaction regions in these 3D structures and we offer insights on the missing regions in crystal structure of the auto-inhibited PAK1. Understanding and modulation of PAK intermolecular interactions can pave the way for PAK blockers and biosensors.

SUBMITTER: Jha RK

PROVIDER: S-EPMC3490965 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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3D structure analysis of PAKs: A clue to the rational design for affinity reagents and blockers.

Jha Ramesh K RK Strauss Charlie E M CE

Cellular logistics 20120401 2

The p21-activated kinase (PAK) family plays a versatile role in cell signaling by forming a hub of interactions. PAKs bind the GTPases like RAC and CDC42. Their proline-rich motifs bind SH3 adaptor proteins such as PIX and NCK. PAKs display nuclear localization signal sites and a potential Integrin binding site. No fully complete structure of the PAKs has been published; partial 3D structures of the PAK family kinases include portions of the auto-inhibited PAK1, GTPase bound to small peptides fr ...[more]

PMID: 23162739

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Project description:Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D?=??1?-?5??M. Beyond the affinity threshold at K D?<?1??M we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.

Dataset Information

3D structure analysis of PAKs: A clue to the rational design for affinity reagents and blockers.

Publications

3D structure analysis of PAKs: A clue to the rational design for affinity reagents and blockers.

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