Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP5_1–486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP5_1–486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Guanylate binding proteins (GBPs) are a family of interferon (IFN)-inducible guanosine triphosphatases (GTPases) that play important roles in innate immunity against diverse intracellular pathogens (1). Many GBPs show activities against bacterial and protozoan pathogens, such as Toxoplasma gondii, Chlamydia trachomatis, Legionella, and Mycobacterium tuberculosis (2). Some of them also have antiviral functions (3). Recently, human GBP5 (hGBP5) was found to restrict HIV-1 by interfering with the processing and incorporation of the viral envelope glycoprotein (Env) (4). A follow-up study revealed that hGBP5 and its paralogue hGBP2 suppress the activity of the virus-dependency factor furin, thereby inhibiting the proteolytic processing of the immature Env precursor gp160 into mature gp120 and gp41 required for virion infectivity (5). Furin is critical for proteolytic cleavage of many viral envelope proteins (6). In support of a key role in innate antiviral immunity, hGBP2 and hGBP5 also restrict other furin-dependent viruses, such as measles, Zika, and highly pathogenic avian influenza A viruses (5).GBPs belong to the dynamin superfamily of large GTPases (7). These are characterized by an N-terminal large GTPase domain (LG domain) and one or more stalk domains (8), usually involved in oligomerization. The stalk domain of GBPs, which is also called C-terminal helical domain (CTHD), comprises the middle domain (MD) and GTPase effector domain (GED). It was proposed that GBPs undergo conformational changes and/or oligomerization upon GTP binding and hydrolysis (9), which may be important for their innate immune functions. Furthermore, GBP1, GBP2, and GBP5 are isoprenylated, and their membrane-binding abilities are modulated by the nucleotide state (10).Despite the importance of this protein family in innate immunity and decades of research, their oligomerization mechanisms remain elusive due to limited structural data (11). The crystal structure of full-length human GBP1 (hGBP1^FL) was determined in its monomeric state (12). The crystal structure of the LG domain alone showed that it is able to form a dimer upon GTP binding (13). Based on these structures, a model of hGBP1^FL in the nucleotide-bound dimeric state was proposed, where the stalk domains protrude to the opposite direction, resulting in an “open” conformation (13). However, the accuracy of this model remains to be tested. Hence, the structures of full-length GBPs in their oligomeric state are in high demand to reveal the detailed molecular mechanisms of GBPs during innate immune responses.Here, we report the crystal structures of hGBP5_1–486 in both its nucleotide-free monomeric state and nucleotide-bound dimeric state, as well as full-length, nucleotide-free human GBP2 (hGBP2^FL). The structures of hGBP5_1–486 and hGBP2^FL are similar to that of hGBP1^FL in the absence of nucleotide. Upon nucleotide binding, however, the stalk domain of hGBP5 undergoes a drastic movement relative to the dimerized LG domain, resulting in a “closed” conformation entirely different from the previously proposed model. Two MD form a hydrophobic interface. Disrupting this interface or mutating the hinge region connecting LG domain and MD, reduces the anti–HIV-1 activity of hGBP2/5, suggesting a crucial role of the closed conformation in their antiviral function. Although the immune functions of the GBP family members are diverse and require specific signals, this dimerization mode is probably shared by all members of the family as revealed by small-angle X-ray scattering (SAXS). On these grounds, we propose a GTP-induced dimerization mechanism of GBPs which lays the foundation to understand the molecular bases of this important innate immune protein family.