CART (Cocaine- and Amphetamine-Regulated Transcript) is a neuropeptide found primarily in the hypothalamus of the brain, where it plays an important role in regulating appetite and energy balance. CART expression increases after cocaine or amphetamine exposure, but it is now best known as an anorectic (appetite-suppressing) peptide regulated by the hormone leptin. Injections of CART into the brain reduce normal feeding, starvation-induced feeding, and block the strong feeding response caused by neuropeptide Y (NPY).
The biologically active region of CART is a small, disulfide-rich C-terminal fragment (residues 48–89), whose three-dimensional structure reveals a compact scaffold stabilized by three disulfide bonds.
Biological Background
CART is synthesized as a larger precursor peptide that undergoes multiple processing steps. In humans, cleavage at a Lys-Arg site produces two major fragments:
CART(1–39) — N-terminal fragment
CART(42–89) — C-terminal fragment
The C-terminal fragment contains six cysteine residues that form three disulfide bonds. This C-terminal piece is highly conserved across species and is the minimal biologically active form capable of suppressing feeding in rodents, fish, and other animals.
Although the biologically active C-terminal peptide is CART(42–89), residues 42–47 do not form any stable structure. In the NMR experiments, these residues were completely disordered and produced no interpretable distance restraints. The first residue that participates in the folded, disulfide-stabilized core is residue 48. Therefore the structurally well-defined region is CART(48–89).
CART is produced in hypothalamic nuclei that regulate appetite. Its levels rise in response to leptin, the hormone produced by adipose tissue that signals sufficient energy stores. CART belongs to a growing family of neuropeptides that modulate hunger, satiety, and energy expenditure.
Structural Description
Overall fold
The structure of CART(48–89) reveals a compact mini-protein dominated by loops and turns, held together by a tight network of three disulfide bonds. This architecture results in an unusually stable fold for such a short peptide.
The three disulfide bonds (connecting half-cystines at positions 55–61, 73–75, and 81–88) create a rigid scaffold similar to a cystine-knot. This fold allows the peptide to maintain a stable three-dimensional shape even in harsh environments.
Secondary structure elements
Although small, CART contains identifiable structural motifs:
A short antiparallel β-sheet between residues 62–64 and 69–71
A second tiny β-sheet between residues 81–82 and 87–88
Three turns: Type I turn (residues 56–59); Type II′ turn (residues 65–68); Type II turn (residues 76–79)
These structural features combine with the disulfide network to produce a compact, globular fold.
Core packing
The peptide’s interior is tightly packed around the disulfide bridges. Ala62 lies at the center of this core, surrounded by hydrophobic side chains. All six cysteine residues are buried, consistent with the need to protect the disulfide knot.
Functional Surface Features
Although the core is rigid, the surface contains distinct regions that likely contribute to biological function.
1. Positively charged patch
A cluster of basic residues forms a prominent charged surface:
Arg64
Lys65
Arg68
Lys71
This positively charged region is strikingly surface-exposed and may interact with negatively charged receptor surfaces, enhance solubility, and help the peptide associate with membranes.
NMR data reveal that Lys71 is unusually well restrained, suggesting it forms a structurally anchored point within this charged pocket..
2. Hydrophobic hotspot
On the opposite face of the molecule lies a contiguous hydrophobic surface composed of:
Phe84
Leu85
Leu86
Leu89
Hydrophobic patches such as this are common recognition elements in peptide–protein interactions and represent a plausible primary receptor-binding interface.
Its high degree of conservation strengthens the hypothesis that this region plays a key functional role.
The presence of two contrasting surface features—a charged patch and a hydrophobic hotspot—suggests that CART likely engages its receptor through a bipartite binding interface involving both electrostatic and hydrophobic contacts.
Structure–Function Insights
Only the C-terminal fragment is active
NMR measurements showed that residues 48–89 fold into a compact, ordered structure, whereas the N-terminal half of the precursor (including residues 42–47) is disordered and produces no long-range NOEs, indicating a lack of structure. This explains why:
CART(1–39) and other N-terminal fragments have no satiety function.
CART(42–89) and CART(48–89) are the only physiologically active forms.
Biological data confirm that only fragments containing the triple-disulfide core show anorectic activity after intracerebroventricular injection.
Stability from disulfide bonds
CART contains six cysteine residues (Cys55, Cys61, Cys73, Cys75, Cys81, Cys88), paired in the unusual pattern:
I–III (Cys55–Cys73)
II–V (Cys61–Cys81)
IV–VI (Cys75–Cys88)
This I–III, II–V, IV–VI pattern is different from classical knottins, but still produces a homologous cystine knot fold. The disulfide pattern locks the peptide into one dominant conformation, shields the hydrophobic core, allows the peptide to be resistant to denaturation and proteolysis, and maintains a consistent receptor-binding surface.
Loops determine specificity
Although the core is highly rigid, the surrounding loops differ in length and shape compared to classical knottins:
Loop 64–69 is the largest among the seven related peptides in the structural comparison.
Loop 84–86 is the shortest among the aligned knottin structures.
These loops show fewer NOEs and higher dihedral-angle variability, are partially flexible, and likely encode functional specificity because the rigid core provides shape, but the loops determine exact receptor interactions.
Thus, CART preserves a knottin-like fold but modifies loop architecture to achieve its own biological function.
Distinct surface patches suggest possible receptor-binding interfaces
The solved structure reveals two major surface features:
A. A large positively charged cluster
Formed by Arg64, Lys65, Arg68, and importantly Lys71, which is structurally restrained (has extensive NOEs, indicating a fixed position rather than a floppy side chain).
This charged patch may interact with negatively charged surfaces on a receptor, help orient the peptide relative to the membrane, or contribute to solubility and targeting.
Lys71 specifically forms a positive “pocket” that may represent a binding hotspot.
B. A hydrophobic hotspot
A continuous hydrophobic surface is formed by Phe84, Leu85, Leu86, Leu89 (and smaller hydrophobic neighbors like Pro53, Val52, Val63, Leu72).
This hydrophobic patch is large and contiguous, remains solvent-exposed despite being hydrophobic, and is a strong candidate for the primary receptor-binding interface.
Therefore, area is a possible hot spot for receptor interaction due to its size and conservation.
Structural features correlate with biological activity
Fragments shorter than CART(48–89)—such as CART(49–63) in goldfish—show reduced anorectic activity (page 6) because they contain parts of the β-hairpin (residues 62–64, 69–71), but lack the full disulfide knot and hydrophobic patch.
This supports the idea that the full cystine knot is required for stable receptor binding, while the hydrophobic and charged surfaces are key functional epitopes.
Thus, the structure directly explains why only certain cleavage products have physiological potency.
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