An allosteric hot spot in the tandem SH2 domain of ZAP‐70 regulates T‐cell signaling

Abstract

The T‐Cell signaling commences with the binding of Antigen Presenting Cell with T‐cell Receptor (TCR) Complex leading to the recruitment of a non‐receptor tyrosine kinase, Zeta Associated Protein‐70 (ZAP‐70), to the membrane. ZAP‐70 is comprised of the tandem SH2 domain (tSH2), which is made up of N‐terminal SH2 (N‐SH2) and C‐terminal SH2 (C‐SH2) domains, and a carboxy‐terminal kinase domain. The two SH2 domains cooperatively bind to the doubly‐phosphorylated ITAM (ITAM‐Y2P) motif resulting in a conformational rearrangement in the whole of the regulatory module, thereby releasing the autoinhibitory conformation of the kinase domain. However, the structural mechanism of how the two SH2 domain cross‐talk during ITAM‐Y2P binding and dissociation from the membrane are poorly understood. We observed that the tSH2 domain undergoes a biphasic structural transition while binding to the doubly‐phosphorylated ITAM‐ ζ1 (ITAM‐Y2P‐ ζ1) peptide. The C‐SH2 binds first to one of the phosphotyrosine in ITAM‐Y2P motifs, leading to the formation of an encounter complex which further leads to the binding by N‐SH2 domain. NMR titration and Network analysis studies show that an intra‐residue network, which allosterically interconnects the N‐SH2 and C‐SH2 binding sites, plays a central role in ITAM‐Y2P binding. Further, a mutation in the allosteric network (F117A, W165C) uncouples the two binding sites, causing autoimmune arthritis in SKG mice. The allosteric coupling in ZAP‐70 is found to be unique and thus plays a significant role in T‐cell regulation.Support or Funding InformationAuthors are thankful to Prof. John Kuriyan and Prof. David E. Wemmer for access to the 900 MHz NMR spectrometer at the University of California, Berkeley. Dr. Jeffrey G. Pelton and Dr. Patrick R. Visperas at the University of California, Berkeley for NMR data collection and sample preparation. Authors thank Prof. Gautam Basu and Mr. Barun Majumder at Bose Institute, India for access to 700 MHz NMR spectrometer. Authors are thankful to Dr. Ashima Bhattacharjee, Dr. Pradip K. Tarafdar, and Prof. Pradipta Purkayastha for access to ITC and fluorimeter. Authors thank Prof. Giuseppe Melacini and Prof. Maitrayee DasGupta for helpful discussion. The authors thanks research funding from IISER Kolkata, infrastructural facilities supported by IISER Kolkata and DST‐FIST (SR/FST/LS‐II/2017/93(c)). This work is supported by grant from SERB (ECR/2015/000142) and DBT Ramalingaswami Fellowship (BT/RFF/Re‐entry/14/2014) to Rahul Das.Figure 1