Evolutionary, Structural and Biochemical Evidence for a New Interaction Site of the Leptin Obesity Protein

Corresponding author: Eric A. Gaucher, Department of Chemistry, 440 Leigh Hall, University of Florida, Gainesville, FL 32611-7200., gaucher{at}ufl.edu (E-mail)

Communicating editor: Y.-X. FU

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

The Leptin protein is central to the regulation of energy metabolism in mammals. By integrating evolutionary, structural, and biochemical information, a surface segment, outside of its known receptor contacts, is predicted as a second interaction site that may help to further define its roles in energy balance and its functional differences between humans and other mammals.

THE Leptin protein has been a focus of energy metabolism and obesity studies since its discovery in obese mice that are lacking a functional leptin gene (ZHANG et al. 1994 ; FRIEDMAN and HALAAS 1998 ; MANTZOROS 1999 ). In these mice, as in several other mammalian species, injections of exogenous Leptin lead to significant weight loss, a result that has generated much interest in the hormone as a potential antiobesity drug (FRIEDMAN 1998 ). However, most obese human patients show an excess of Leptin, rather than deficiency, thereby implying that its detailed actions in energy metabolism are different in us vs. other mammals (CHICUREL 2000 ; HOFBAUER and HUPPERTZ 2002 ). This possibility is bolstered by evolutionary studies that provide evidence of positive (adaptive) selection on the Leptin of hominoids (humans and great apes; BENNER et al. 2002 ; SILTBERG and LIBERLES 2002 ).

To better understand these functional differences, we extended these earlier comparative studies by integrating our evolutionary results with the available structural and biochemical information for Leptin. The same multiple alignment and phylogeny for the coding DNA sequences of mature Leptin (146 residues), as used before (BENNER et al. 2002 ; SILTBERG and LIBERLES 2002 ), were analyzed with PAML (YANG 1997 ). This evolutionary analysis included the estimation of the nonsynonymous (NS) to synonymous (SYN) rate ratios (amino acid vs. silent substitution rates or ) and the reconstruction of ancestors and their inferred substitutions. The amino acid replacements for the NS substitutions were then mapped onto the known tertiary structure of human Leptin and evaluated against the functional evidence that a specific segment of this protein is primarily responsible for appetite suppression and weight loss in obese mice (ZHANG et al. 1997 ; GRASSO et al. 1999A , GRASSO et al. 1999B ).

Starting with free to vary across branches, an evolutionary model with different transition vs. transversion rates and the same but unequal base frequencies for all three codon positions was selected by the likelihood ratio tests (LRTs; Table 1; HUELSENBECK and RANNALA 1997 ). Given this model, was estimated as <1 for all branches with more than five inferred substitutions, except for the hominoid stem (Fig 1). Values of < 1, = 1, and > 1 are indicative of negative selection, neutral evolution, and positive selection, respectively (YANG and BIELAWSKI 2000 ). Thus, the stem hominoid with = 1.66 corresponds to the most likely episode of positive selection, underlying the known functional differences between human and mouse (BENNER et al. 2002 ; SILTBERG and LIBERLES 2002 ). This hypothesis is corroborated by the significant increase of NS to SYN substitutions in the stem hominoid relative to all other mammals and to its immediate primate ancestor and descendant hominoid clade (Table 2A and Table 2B). More importantly, it is congruent with available structural and biochemical information for mammalian Leptin (see below).

View larger version (35K): In this window In a new window Download PPT slide   Figure 1. Accepted phylogeny following earlier evolutionary studies of leptin (BENNER et al. 2002 ; SILTBERG and LIBERLES 2002 ). Common names and SWISSPROT accession numbers are given in brackets, and three key periods of primate evolution are labeled I, II, and III (Table 2B). Values of , as calculated with and without segment 85–119, are presented in that order for each branch, with boldface type highlighting those estimates based on more than five substitutions. Branch lengths, parameter estimations, and other calculations (Fig 2B) were determined with PAML (YANG 1997 ). However, as in the earlier evolutionary studies, the available chicken and turkey leptins were not included in this analysis because of persistent concerns about their authenticity (FRIEDMAN-EINAT et al. 1999 ; DOYON et al. 2001 ). Nevertheless, identical to near-identical results were obtained when these bird sequences were included (results not shown).

The administration of synthetic Leptin peptides identifies positions 85–119 as critical for appetite suppression and weight loss in obese mice (GRASSO et al. 1999A , GRASSO et al. 1999B ). Segment 85–119 includes the end of -helix C and intervening C/D loop with helix E (Fig 2A) and is outside the region where Leptin contacts its receptor (interface of -helices A and C; HIROIKE et al. 2000 ). Removal of segment 85–119 from the evolutionary analysis reduced for the stem hominoid from 1.66 to 0.52 (Fig 1). Of the 11 substitutions for this stem, 5 NS and no SYN changes mapped to this segment, which was significant (Table 2C). Furthermore, segment 85–119 packs onto the folded protein core by hydrophobic interactions between helix E and residues 60, 64, and 68 (ZHANG et al. 1997 ). As 2 NS substitutions of the stem hominoid mapped to residues 60 and 68, 7 of its 9 NS changes were thereby associated either directly or indirectly with segment 85–119 (Fig 2). In contrast, no NS substitution of the stem hominoid was directly associated with the Leptin-receptor binding domain.

View larger version (65K): In this window In a new window Download PPT slide   Figure 2. (A) Tertiary structure of human Leptin, PDB accession 1AX8 (ZHANG et al. 1997 ), as rendered with MOLSCRIPT (KRAULIS 1991 ). (B) Inferred replacements for the eight sites with NS substitutions in the stem hominoid (Fig 1). The posterior probabilities for the reconstructed codons of the primate (I) vs. hominoid (II) ancestors are given in parentheses. These same replacements, ancestral reconstructions, and posterior probabilities are also obtained by the other evolutionary models in Table 1 and with the addition of the bird leptins to the analysis (results not shown).

The evolutionary, structural, and biochemical information implicates segment 85–119 as of special functional significance. The physicochemical properties and finer structural details of its conserved residues now point to a more specific function for this segment. Fourteen of its 15 conserved positions are fixed for charged and strongly hydrophobic residues (Fig 3). This mix of charged and hydrophobic residues, with their outwardly projecting side chains, predicts a second binding site for Leptin-protein interactions, which is separate from that for its receptor (Fig 2; BENNER and GERLOFF 1991 ). At least some of the six positions with NS substitutions in the stem hominoid, which are directly or indirectly related to segment 85–119, may then contribute new hydrophobic, charged, and smaller residues that may alter the secondary structure and specific binding properties of this second interaction site. For example, the conserved G118 of hominoids permits a more pronounced turn at the N terminus of this segment relative to that predicted for L118 of the other mammals. In these ways, segment 85–119 may underlie the functional differences between human and other nonhominoid Leptins (e.g., why this hormone is central to energy expenditure in mice, but apparently not in us; MANTZOROS 1999 ; HOFBAUER and HUPPERTZ 2002 ).

View larger version (82K): In this window In a new window Download PPT slide   Figure 3. Multiple Leptin alignment. Asterisks highlight conserved residues and arrows mark those sites with the NS substitutions of the stem hominoid (Fig 1). The two SYN substitutions of the stem hominoid occur at residues 37 and 121. Segment 85–119 is shaded, whereas helices A–E are labeled (Fig 2).

This integrative study of Leptin calls for new experiments for the greater understanding of its roles in the energy metabolism of humans and other mammals (FRIEDMAN and HALAAS 1998 ; MANTZOROS 1999 ). The prediction of a new binding site, separate from that for its receptor, argues for experimental assays of Leptin-protein interactions (e.g., with yeast two-hybrid systems; VON MERING et al. 2002 ). Such experiments can test whether the regulation of metabolic rate vs. feeding depends on the same or different protein-protein interactions and domains of Leptin, while documenting further its specific roles in energy metabolism (GRASSO et al. 1999A , GRASSO et al. 1999B ). Furthermore, the inferred amino acid replacements of the stem hominoid supplement the known mutations in human and mouse Leptin and thereby offer additional targets for site-directed mutagenesis of its function (VERPLOEGEN et al. 1997 ). Finally, as mammalian Leptin remains largely under purifying selection outside of hominoids, it may still prove beneficial to develop it as a weight control drug for domesticated animals (HOSSNER 1998 ).

	ACKNOWLEDGMENTS

We thank M. R. Tennant, Z. Yang, and F. Zhang for their comments. This study was supported in part by a National Research Council and NASA Astrobiology Institute postdoctoral fellowship (E.A.G.), by funds from the Department of Zoology, University of Florida, and by NASA Exobiology grant NAG5-9030 (S.A.B.).

Manuscript received July 11, 2002; Accepted for publication January 7, 2003.

	LITERATURE CITED

TOP ABSTRACT LITERATURE CITED

BENNER, S. A. and D. GERLOFF, 1991  Patterns of divergence in homologous proteins as indicators of secondary and tertiary structure: a prediction of the structure of the catalytic domain of protein-kinases. Adv. Enzyme Regul. 31:121-181.[Medline]

BENNER, S. A., M. D. CARACO, J. M. THOMSON, and E. A. GAUCHER, 2002  Planetary biology: paleontological, geological, and molecular histories of life. Science 296:864-868.[Abstract/Free Full Text]

CHICUREL, M., 2000  Whatever happened to Leptin? Nature 404:538-540.[Medline]

DOYON, C., G. DROUIN, V. L. TRUDEAU, and T. W. MOON, 2001  Molecular evolution of Leptin. Gen. Comp. Endocrinol. 124:188-198.[Medline]

FRIEDMAN, J. M., 1998  Leptin, Leptin receptors, and the control of body weight. Nutr. Rev. 56(II):S38-S46.[Medline]

FRIEDMAN, J. M. and J. L. HALAAS, 1998  Leptin and the regulation of body weight in mammals. Nature 395:763-770.[Medline]

FRIEDMAN-EINAT, M., T. BOSWELL, G. HOREV, G. GIRISHVARMA, and I. C. DUNN et al., 1999  The chicken leptin gene: Has it been cloned? Gen. Comp. Endocrinol. 115:354-363.[Medline]

GRASSO, P., M. C. LEINUNG, and D. W. LEE, 1999a  Epitope mapping of secreted mouse leptin utilizing peripherally administered synthetic peptides. Regul. Pept. 85:93-100.[Medline]

GRASSO, P., D. W. WHITE, L. A. TARTAGLIA, M. C. LEINUNG, and D. W. LEE, 1999b  Inhibitory effects of Leptin-related synthetic peptide 116–130 on food intake and body weight gain in female C57BL/6J ob/ob mice may not be mediated by peptide activation of the long isoform of the Leptin receptor. Diabetes 48:2204-2209.[Abstract]

HIROIKE, T., J. HIGO, H. JINGAMI, and H. TOH, 2000  Homology modeling of human Leptin/Leptin receptor complex. Biochem. Biophys. Res. Commun. 275:154-158.[Medline]

HOFBAUER, K. G. and C. HUPPERTZ, 2002  Pharmacotherapy and evolution. Trends Ecol. Evol. 17:328-334.

HOSSNER, K. L., 1998  Cellular, molecular and physiological aspects of Leptin: potential application in animal production. Can. J. Anim. Sci. 78:463-472.

HUELSENBECK, J. P. and B. RANNALA, 1997  Phylogenetic methods come of age: testing hypotheses in an evolutionary context. Science 276:227-232.[Abstract/Free Full Text]

KRAULIS, P. J., 1991  MOLSCRIPT: a program to produce both detailed and schematic plots of proteins structures. J. Appl. Crystallogr. 24:946-950.

MANTZOROS, C. S., 1999  The role of Leptin in human obesity and disease: a review of current evidence. Ann. Intern. Med. 130:671-680.[Abstract/Free Full Text]

NEI, M., and S. KUMAR, 2000 Molecular Evolution and Phylogenetics. Oxford University Press, New York.

SILTBERG, J. and D. A. LIBERLES, 2002  A simple covarion-based approach to analyze nucleotide substitution rate. J. Evol. Biol. 15:588-594.

VERPLOEGEN, S. A. B. W., G. PLAETINCK, R. DEVOS, J. VAN DER HEYDEN, and Y. GUISEZ, 1997  A human Leptin mutant induces weight gain in normal mice. FEBS Lett. 405:237-240.[Medline]

VON MERING, C., R. KRAUSE, B. SNEL, M. CORNELL, and S. G. OLIVER et al., 2002  Comparative assessment of large-scale data sets of protein–protein interactions. Nature 417:399-403.[Medline]

YANG, Z. and J. P. BIELAWSKI, 2000  Statistical methods for detecting molecular adaptation. Trends Ecol. Evol. 15:496-503.[Medline]

YANG, Z. H., 1997  PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13:555-556.[Free Full Text]

ZHANG, F., M. B. BASINSKI, J. M. BEALS, S. L. BRIGGS, and L. M. CHURGAY et al., 1997  Crystal structure of the obese protein Leptin-E100. Nature 387:206-209.[Medline]

ZHANG, Y., R. PROENCA, M. MAFFEI, M. BARONE, and L. LEOPOLD et al., 1994  Positional cloning of the mouse obese gene and its human homologue. Nature 372:425-432.[Medline]

This article has been cited by other articles:

				R. N. Trotter-Mayo and M. R. Roberts Leptin Acts in the Periphery to Protect Thymocytes from Glucocorticoid-Mediated Apoptosis in the Absence of Weight Loss Endocrinology, October 1, 2008; 149(10): 5209 - 5218. [Abstract] [Full Text] [PDF]

				D. A. McClellan, E. J. Palfreyman, M. J. Smith, J. L. Moss, R. G. Christensen, and J. K. Sailsbery Physicochemical Evolution and Molecular Adaptation of the Cetacean and Artiodactyl Cytochrome b Proteins Mol. Biol. Evol., March 1, 2005; 22(3): 437 - 455. [Abstract] [Full Text] [PDF]