Na⁺/H⁺ Exchangers (SLC9As) are of pivotal importance in several physiological processes, and their dysfunction is linked to the pathogenesis of several diseases. The Na⁺/H⁺ Exchanger 1 (NHE1) is ubiquitously expressed at the plasma membrane. It contains two domains, the transmembrane ion translocation domain and a large cytoplasmic regulatory domain. Despite its physiological and pathophysiological importance, high resolution structural data of NHE1 is missing, and even though several binding partners and phosphorylation sites of the NHE1 regulatory domain are known and many more suggested, the molecular structures and mechanisms underlying NHE1 regulation and function are still incompletely understood.
In this work, in silico analysis revealed two structured and two intrinsically disordered regions in the regulatory domain. A divide-and-conquer approach was used to identify subdomains that are potentially suitable for structural characterization of the regulatory domain. Further, a novel putative dimerization site was found within a helical region of the regulatory domain. The lipid binding region located within the membrane proximal part of the regulatory domain was further examined and two structural elements, which are formed upon membrane interaction, were identified. 
The importance of intrinsic disorder in regulation of the scaffolding function of NHE1 was characterized for the first time. Three sites in the disordered distal part of the NHE1 regulatory domain were identified to interact with the extracellular regulated protein kinase 1 (ERK2), specifically one D-domain, a non-canonical F-site, and the substrate-site T_{779P. This low-affinity, non-cooperative tri-partite interaction we term a Shuffle complex. Evidence is provided for cross-regulation of NHE1 and ERK2, specifically direct phosphorylation of six sites in NHE1 by active ERK2 in vitro, and modulation of ERK1/2 activity by NHE1 in vivo. Phosphorylation of NHE1 by active ERK2 was found to be distributive, leading to the identification of primary and secondary sites. The structural and functional consequences of NHE1 phosphorylation are still poorly understood. Here, we could show that NHE1 phosphorylation by active ERK2 has a strong stabilizing effect on two transient helices in the disordered distal region of the NHE1 regulatory domain.} 
In conclusion, this work provides several new contributions to the structural and functional understanding of NHE1