Design, Pharmacology, and NMR Structure of a Minimized Cystine Knot with Agouti-Related Protein Activity

Abstract

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The NMR structure of this active domain was recently determined and suggested that melanocortin receptor contacts were made primarily by two loops presented by a well-structured cystine knot domain within AGRP(87-132) [McNulty et al. (2001) Biochemistry 40, 15520-15527]. This hypothesis is tested here with NMR structure and activity studies of a 34-residue AGRP analogue designed to contain only the cystine knot domain. The designed miniprotein folds to a homogeneous product, retains the desired cystine knot architecture, functions as an antagonist, and maintains the melanocortin receptor pharmacological profile of AGRP(87-132). The AGRP-like activity of this molecule supports the hypothesis that indeed the cystine knot region possesses the melanocortin receptor contact points. Moreover, this potent AGRP analogue is synthetically accessible, may serve in the development of therapeutics for the treatment of diseases related to energy balance. and may also find use as a new reagent for probing melanocortin receptor structure and function.

Receptor−Antagonist Interactions in the Complexes of Agouti and Agouti-Related Protein with Human Melanocortin 1 and 4 Receptors,

“…The computations included three steps: (1) comparative model of ASIP(93-132), which demonstrates 50% homology to AGRP(87-132), using a higher quality NMR structure of AGRP(87-120) [1mr0 (8)] as a template for the first 34 residues and a second NMR model of a longer fragment AGRP(87-132) [1hyk (18)] as a template for the C-terminal loop 121-132, (2) homology modeling of MCR1 and MCR4, based on the crystal structure of rhodopsin (1gzm) (26), and (3) calculation of three receptor-antagonist complexes using our mutagenesis data to identify residues important for binding of ASIP(93-132) and AGRP(87-132) by the corresponding receptors.…”

Structures of the Agouti Signaling Protein

“…12 Oxidative folding was performed in a mixture of oxidized and reduced glutathione and low concentrations of guanidinium. 13 A wide series of conditions were explored, however, the best yield relative to pure unfolded peptide was approximately 10%. An analytical HPLC trace of ASIP(80-132) following a folding trial is shown in Figure 1(a).…”

“…This proposal is also consistent with our previous observation that AgRP(87-120), a mini-protein that contains only the central and N-terminal loops, exhibits MCR selectivity and antagonism equivalent to that of the full AgRP C-terminal domain. 13 Alternative conformations of ASIP-YY, dictated by the cis-trans isomerization of the Ala104-Pro105 peptide bond, contain a persistent cis peptide bond at Pro102-Pro103, yet the N-terminal loop of AgRP is devoid of cis Xaa-Pro bonds. As demonstrated by our mutagenesis studies, this apparent paradox is explained because a single cis bond at either position of ASIP-YY yields a protein with significantly reduced activity, while two cis bonds yield a protein with normal activity.…”

Design, Synthesis and Biological Evaluation of Ligands Selective for the Melanocortin-3 Receptor

“…Along with the NMR structure of AGRP (the endogenous antagonist of melanotropin) (PDB: 1HYK) [66] and MTII (potent agonist) [67], the 3D pharmacophore of human melanocortin receptor ligands has been partially deciphered. Results from these biophysical and chemical studies can be crucial for designing novel selective agonist and antagonist melanotropins [52].…”