Assignment 2

Structure

The Leptin protein is encoded by the ob gene on chromosome 7 (127.67-127.68 Mb) of humans. The gene encodes a polypeptide with 167 amino acids and a weight of 16 kDa [2]. The 167 amino acid sequence has a 21 amino acid signal sequence which is cleaved in post translational processing and a 147 amino acid protein circulates in the blood [1].

The secondary structure of the circulating protein is an alpha helix in which the amino acid residues interact by hydrogen bonds. The tertiary structure consists of a helical bundle that consists of four alpha helical domains. These domains are held together by several loop structures that all differ in length [2]. These loop structures allow two of the alpha helices to be antiparallel meaning that their sequences of amino acids run in opposite directions (N-terminals on opposite ends). The other domains run in the same direction so that overall the protein has the first two helical domains pointing upward while the other two domains are pointing downward as visible in the Figure above.


The core of the tertiary structure (helix bundle) contains a region of hydrophobic residues
(Val, Ile, Met, Ala) within two of the helices. These residues interact with leu,val residues contained in the longest loop structure as well as with each other [2]. The hydrophobic interactions between the residues and their position at the top of the bundle contribute to stabilization of the protein core and thus the protein as a whole.

Leptin's structure is most similar to that of the cytokines, particularly Interleukin-6 (Il-6) which can induce or inhibit inflammatory immune responses. The granulocyte colony stimulating factor (G-CSF) is also very similar to leptin in terms of molecular structure. G-CSF is also a cytokine/growth factor that has immune function in the development of bone marrow.

Just as leptin is simlar to the cytokines, its receptor is similar to the cytokine receptors and it is classified under the cytokine receptor family (Class I). These receptors have an immunoglobulin like domain in the middle of two CRH domains as well as two fibronectin domains (type III) [3]. The mechanism of action for the receptors is by Janus Kinase 2 (Jak2)/STAT pathway where hormone binding causes recruitment of Jak2 and phosphorylation of receptro tyrosines. This recruits the transcription factor STAT which is also phoshorylated (activated), binds to certain promoters and activates transcription of specific genes.

Leptin-Leptin receptor interactions
Leptin-receptor interactions involve three binding sites located at different helices in the protein, which was shown by Zabeau et al in an experiment that involved the mapping of leptin- receptor binding sites. The helices of the protein are named as helix A through D and two of the binding were found to be within the helix D. A third binding site involves two helices, A and C which associates with one of the CRH domains on the receptor. The binding sequences located at the end of the D helix associate with the immunoglobulin like region of receptor [3]. A model of leptin- leptin receptor interactions is shown in the figure below.

Leptin interaction with receptor (LR)

Model of Class I cytokine receptor

Leptin protein alignment

The degree of correlation of leptin hormone structure between different closely related species can be important in explaining the various effects of leptin which can differ between species. It is interesting to see how close in molecular structure, a hormone can be (in this case leptin), between different species.

Protein alignments for leptin were obtained using Blast and ClustalW from which alignment scores were obtained between each species selected. A high score indicates a high degree of similarity between sequences. Figure 1 shows the actual protein sequence alignment from which the location of identical residues between the sequences can be seen. The alignment scores in Table 1 shows that leptin hormone is highly conserved among mammals. Overall the alignment scores correlate with the phylogenetic data shown in Figure 2. The human hormone shows a high similarity to the mouse with 82% score while it has the lowest similarity with the rabbit (77%). Conservation of the hormone sequence is also shown by the high alignment score between human and the cat which is on a different evolutionary lineage as shown in Figure 2.

Figure 1: Sequence alignment of leptin hormone in different mammal species produced using ClustalW

Figure 2: Phylogenetic Tree

References

1 Ruffing, R., & Bardot, J. 2007. Leptin E-100, the obese protein. Retrieved October 18, 2008 from the world wide web: http://biology.kenyon.edu/BMB/Chime2/2005/Leptin%20Model/Frames/start.html

2 Wikipedia, 2008. Leptin. Retrieved October 18, 2008 from the world wide web: http://en.wikipedia.org/wiki/Leptin

3 Zabeau, L., Vandekerckhove, J., Tavernier, j., Peelman, F., Iserentant, H., and De Smet A.S. 2006. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin -leptin receptor complex. Journal of Biol. Chem. Jun 2:28/(22): 15496-504