Conformational flexibility of GRASPs and their constituent PDZ subdomains reveals structural basis of their promiscuous interactome

Abstract

The Golgi complex is a central component of the secretory pathway, responsible for several critical cellular functions in eukaryotes. The complex is organized by the Golgi matrix that includes the Golgi Reassembly and Stacking Protein (GRASP), which was shown to be involved in cisternae stacking and lateral linkage in metazoan. GRASPs also have critical roles in other processes, with an unusual ability to interact with several different binding partners. The conserved N-terminus of the GRASP family includes two PDZ domains. Previous crystallographic studies of orthologues suggest that PDZ1 and PDZ2 have similar conformations and secondary structure content. However PDZ1 alone mediates nearly all interactions between GRASPs and their partners. In this work, NMR, Synchrotron-Radiation Circular Dichroism and Molecular Dynamics were used to examine the structure, flexibility and stability of the two constituent PDZ domains. GRASP PDZs are structured in an unusual β3α1β4β5α2β6β1β2 secondary structural arrangement and NMR data indicates that the PDZ1 binding pocket is formed by a stable β2-strand and a more flexible and unstable α2-helix, suggesting an explanation for the higher PDZ1 promiscuity. The conformational free energy profiles of the two PDZ domains were calculated using Molecular Dynamics simulations. The data suggest that, after binding, the protein partner significantly reduces the conformational space that GRASPs can access by stabilizing one particular conformation, in a partner-dependent fashion. The structural flexibility of PDZ1, modulated by PDZ2, and the coupled, coordinated movement between the two PDZs enable GRASPs to interact with multiple partners, allowing them to function as promiscuous, multitasking proteins.

In vivo amyloid-like fibrils produced under stress

“…Our group has been exploring the biophysics of GRASPs in the last few years [20–25]. In one of our last contributions [22], we described novel structural features of Grh1, the GRASP from Saccharomyces cerevisiae .…”

Resurrecting Golgi proteins to grasp Golgi ribbon formation and self-association under stress

“…The strand 2 and the helix 2 form the binding groove of GRASP's PDZs [ 12,45,46 ]. Interestingly, the GRASP promiscuous interactome was previously suggested as a direct outcome of the somewhat low stability and high flexibility of the 2 helix in the PDZ1, a region enriched in high configurational frustration (Figure 2B) [ 25 ].…”

“…It comes as quite a surprise that there is still a significant gap in understanding how GRASPs can perform those functionalities. The second portion of the GRASP's structure, the so-called Serine and Proline-Rich (SPR) domain, has a regulatory function [ 15 ] and presents a very low sequence identity even for closely related species [ 25 ].…”

“…The N-terminal one, called the GRASP domain (DGRASP), is formed by two PDZ subdomains with an unusual circular permutation in eukaryotes, making them resemble prokaryotic PDZs [ 24 ]. The PDZs are connected in tandem to allow rigid body reorganization, facilitating their interaction with multiple different protein partners [ 25 ]. This promiscuity for interaction with several different protein partners and their unusual structural plasticity [ 26,27 ] might be the convergent features of GRASPs in different cell functional tasks, including as anchoring factors of Golgins [ 12,15,23 ], their direct participation in Golgi dynamics during mitotic and apoptotic times [ [28][29][30] and their self-association during stress conditions triggering UPS [ 4,31 ].…”

Myristoylation and its effects on the human Golgi Reassembly and Stacking Protein 55

“…The highly conserved GRASP domain (DGRASP) is myristoylated at the glycine 2 (Gly2) and anchored to the Golgi membranes (Jarvela and Linstedt 2012). DGRASP is, in turn, formed by two PDZ ( P SD95/ D lgA/ Z o-1) sub-domains(Mendes et al 2020), conventionally named PDZ 1 and 2. The interaction between GRASPs and their golgin partners involves the participation of the C-terminal region of the golgin that interacts at two distinctsites in DGRASP concurrently: the canonical peptide-binding groove of the PDZ1 and additional residues located either on the surface of the PDZ2 or in the region connecting both subdomains (Hu et al 2015; Zhao et al 2017).…”