Catalytic activity of human guanylate‐binding protein 1 coupled to the release of structural restraints imposed by the C‐terminal domain

Abstract

Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and c-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG: LG interface. After releasing the cleaved c-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the b-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein.

Human guanylate binding proteins: nanomachines orchestrating host defense

“…Extensive structural and biochemical studies of non-farnesylated GBP1 revealed how GBP1 catalyzes the hydrolysis of GTP in two consecutive steps to GDP and GMP [18,19,[34][35][36][37][38][39][40][41][42][43] (Figure 1A -B). Initial GTP binding by monomeric GBP1 is mediated by the consensus nucleotide binding motifs (G1-4) of its nucleotide binding pocket.…”

“…LG:LG interface between two GBP1 molecules resulting in a head-to-head dimer which is in a rapid equilibrium with GTP-bound monomers [36,41,43].…”

“…Cleavage of the γ-phosphate bond results in substantial conformational changes in the phosphate cap and the phenylalanine patch (F-patch) formed by residues in the LG α3' and α4' helices. These conformational changes disrupt a network of salt bridges formed between residues R227-K228 of the LG helix α4' and four aspartates residing in the GED [37,41] ( Figure 1A).…”

“…The plasticity of the catalytic center for nucleotide binding enables the hydrolysis of GDP to GMP through the same catalytic process as the hydrolysis of GTP to GDP [34,40,41,43,45].…”

“…Subsequent studies using SEC in combination with multi-angle light scattering (MALS) considered changes to protein shape The constitutive open conformation of the GBP5 monomer can therefore be explained by the lack of specific intramolecular regulatory mechanisms present in GBP1, which were shown to promote the safety pin-like conformation of nucleotide-free GBP1. The safety pin-like conformation of nucleotide-free GBP1 monomers is effectuated by the salt bridge network between the LG and the GED, the F-patch, and the additional salt bridge between K62 (switch II) and D255 (guanine cap) [36,37,41,42]. In contrast to GBP5, GBP2 shares with GBP1 a homologous F-patch and most of the residues mediating salt bridge contacts within the LG and between the LG and the GED.…”