Crystal Structure of Visfatin/Pre-B Cell Colony-enhancing Factor 1/Nicotinamide Phosphoribosyltransferase, Free and in Complex with the Anti-cancer Agent FK-866

Abstract

Visfatin/pre-B cell colony-enhancing factor 1 (PBEF)/nicotinamide phosphoribosyltransferase (NAmPRTase) is a multifunctional protein having phosphoribosyltransferase, cytokine and adipokine activities. Originally isolated as a cytokine promoting the differentiation of B cell precursors, it was recently suggested to act as an insulin analog via the insulin receptor. Here, we describe the first crystal structure of visfatin in three different forms: apo and in complex with either nicotinamide mononucleotide (NMN) or the NAmPRTase inhibitor FK-866 which was developed as an anti-cancer agent, interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. The crystal structures of the complexes with either NMN or FK-866 show that the enzymatic active site of visfatin is optimized for nicotinamide binding and that the nicotinamide-binding site is important for inhibition by FK-866. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin and does not share the binding site with insulin on the insulin receptor. To predict binding sites, the potential interaction patches of visfatin and the L1-CR-L2 domain of insulin receptor were generated and analyzed. Although the relationship between the insulin-mimetic property and the enzymatic function of visfatin has not been clearly established, our structures raise the intriguing possibility that the glucose metabolism and the NAD biosynthesis are linked by visfatin.

Introduction

The recently identified adipokine visfatin, which is highly enriched in the visceral fat of humans,¹ corresponds to pre-B cell colony-enhancing factor 1 (PBEF), which has been described as both a secreted cytokine playing a highly conserved role in innate immunity and an intracellular phosphoribosyltransferase (PRTase). The novel visfatin gene was first isolated from a human peripheral blood lymphocyte cDNA library² and encodes a secreted 52 kDa cytokine that acts as a growth factor for early stage B cells, even though it lacks a signal peptide. But subsequent experiments revealed additional biological activities that suggested intracellular as well as extracellular localization of visfatin. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin (K_d = 3 nM). Visfatin does not compete with insulin, however, suggesting that the two proteins bind to separate sites on the receptor.¹

Visfatin is up-regulated in neutrophils and monocytes in response to a variety of inflammatory stimuli and functions as a novel inhibitor of apoptosis. Several lines of evidence suggest the anti-apoptotic activity of visfatin is dependent on its secretion from neutrophils,³ and it appears to be one of the mediators of the later stages of the systemic inflammatory response. Visfatin is also constitutively expressed by the fetal membranes during pregnancy and is up-regulated when the membranes are infected,⁴ suggesting that it may play a role in infection-induced preterm birth.

Other researchers have found visfatin to be primarily situated in the cell nucleus and cytoplasm. Its possible role in NAD biosynthesis is suggested by the discovery that a bacterial homolog is NadV⁵ which has the enzymatic activity of nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyzes the reaction between nicotinamide (NAm) and phosphoribosyl pyrophosphate (PRPP) to yield nicotinamide mononucleotide (NMN) (Figure 1).

NAD can also be synthesized from quinolinic acid (QA) and nicotinate (NA) via analogous pathways. Indeed, Rongvaux et al. characterized murine visfatin as an intracellular NAmPRTase and demonstrated that it can rescue a mutant bacterium deficient in this enzymatic activity. Visfatin also was identified as a positive regulator of NAD-dependent protein deacetylase activity, which is essential for maturation of smooth muscle cells.⁶ Kitani et al. concluded that visfatin is an intracellular protein associated with the cell cycle based on the observation that rat visfatin is localized in the nuclei of confluent cells but in the cytoplasm of proliferating ones.⁷

Notably, visfatin was also found to be the most highly up-regulated gene in a canine model of acute lung injury, thus making visfatin a potentially novel biomarker in acute lung injury.⁸ Visfatin also was identified as one of the genes overexpressed in primary colorectal cancer.⁹ In that regard, FK-866 (Figure 1), which was developed as an anti-cancer agent,¹⁰ interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. Inhibition of NAD synthesis using FK-866 induced apoptosis in human leukemia and hepatocarcinoma cells and in various types of tumor xenografts. Moreover, FK-866 has anti-tumoral, anti-metastatic and strong anti-angiogenic activities in RENCA mice,¹¹ and effectively induced delayed apoptotic cell death in HepG2 human liver carcinoma cells with an IC₅₀ of approximately 1 nM.¹⁰

To gain insight into the mechanism of action by this multifunctional protein, we have determined the first crystal structure of rat visfatin in three forms: the apo form, the nicotinamide mononucleotide (NMN)-bound form and the FK-866-inhibited form. We found that visfatin forms a homodimer and that FK-866 binds at the interface between the two subunits, partially overlapping the NMN binding site. In addition, we carried out a kinetic study using isothermal titration calorimetry (ITC) to assess FK-866 affinity and enzyme substrate specificity, and structural modeling for the visfatin and insulin receptor complex. Analysis of the structures and biochemical data reveal the enzymatic specificity of visfatin, and suggest the model of visfatin and insulin receptor complex.