AbstractLeptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin (13C/15N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core

Becker, Gerald W

Churgay, Lisa M

Hale, John E

Kline, Allen D

Landen, Bryan E

Martin, Debra K

Muth, William L

Rathnachalam, Radhakrishnan

Richardson, John M

Schoner, Brigitte

Ulmer, Maverick

Elsevier - Publisher Connector 

FEBS 18498 FEBS Letters 407 (1997) 239-242 
Leptin is a four-helix bundle: secondary structure by NMR 
Allen D. Kline*, Gerald W. Becker, Lisa M. Churgay, Bryan E. Landen, Debra K. Martin, 
William L. Muth, Radhakrishnan Rathnachalam, John M. Richardson, Brigitte Schoner, 
Maverick Ulmer, John E. Hale 
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 48285-0403, USA 
Received 19 February 1997 
Abstract Leptin is a signaling protein that in its mutant forms 
has been associated with obesity and Type II diabetes. The lack 
of sequence similarity has precluded analogies based on 
structural resemblance to known systems. Backbone NMR 
signals for mouse leptin (13C/1BN -labeled) have been assigned 
and its secondary structure reveals it to be a four-helix bundle 
cytokine. Helix lengths and disulfide pattern are in agreement 
with leptin as a member of the short-helix cytokine family. A 
three-dimensional model was built verifying the mechanical 
consistency of the identified elements with a short-helix cytokine 
core. 
© 1997 Federation of European Biochemical Societies. 
Key words: Nuclear magnetic resonance; Leptin; Obesity; 
Secondary structure; Cytokine-fold 
1. Introduction 
The obese gene encodes a 167-amino-acid polypeptide [1] 
that appears to function in the maintenance of body weight. 
The product of this gene, leptin, is a secreted 146-residue 
protein containing a single disulfide bond. Injection of murine 
or human leptin into ob/ob mice leads to weight loss and 
correction of the diabetic state of these animals [2-4]. Leptin 
appears to act on receptors in the arcuate nucleus to suppress 
neuropeptide Y release [5]. The leptin receptor and various 
splice variants were cloned [6-8] and found to have sequence 
similarity to the class I cytokine receptor family [6]. Interest-
ingly, the expression of the leptin receptor seems to occur in 
many different tissues, and recent findings have implicated 
leptin in reproduction [9] and hematopoiesis [10]. 
Leptin's role as a circulating hormone and the similarity of 
its receptor to cytokine receptors implicate it as a cytokine; 
however, it has no strong sequence similarity with any other 
protein, making modeling of the protein structure difficult. 
Threading analysis of the leptin sequence against a 3-dimen-
sional database indicated that it is compatible with a cytokine 
folding pattern [11]. However, the predicted helical composi-
tion of 39% is considerably lower than the 52% estimated by 
CD measurements [12]. While these studies implicate this fold, 
they do not help to resolve the structural subfamily to which 
leptin belongs. In this paper we will use NMR methodologies 
to determine the exact secondary structure for mouse leptin, 
giving direct experimental verification to its adoption of a 
*Corresponding author. Fax: (317) 276-9722. 
E-mail: a.kline@lilly.com 
Abbreviations: CD, circular dichroism; CSI, chemical shift index; IL-
6, interleukin-6; NMR, nuclear magnetic resonance; NOE, nuclear 
Overhauser effect 
cytokine fold and its similarity to the short-helix subfamily 
of cytokine folds. 
2. Materials and methods 
E. coli expressing murine leptin was grown at 37°C overnight in 
either a modified M-9-defined medium enriched with 15N or in me-
dium (Isotec) enriched in 15N or 13C/15N prepared according to the 
manufacturer's instructions. Inclusion bodies containing leptin were 
isolated by differential centrifugation and the protein was purified and 
renatured as described [5]. The isotopic enrichment of the purified 
protein was estimated by electrospray ionization mass spectrometry 
(PE-Aciex, API-Ill). Prior to NMR studies, the protein was dialyzed 
against a 10% 2H20 solution containing 20 mM PO4 and 1 mM 
EDTA, pH 6.6, then concentrated approximately to 10 mg/ml. 
NMR spectroscopy was performed on a Unity-500 (Varian Instru-
ments) equipped with gradients. Probe temperature was regulated at 
30°C. The following experiments were collected: HSQC(15N), NO-
ESY-HSQC(15N), TOCSY-HSQC(15N), HNCA [13], HNCACB [13], 
CBCA(CO)NNH [13,14], HBHA(CBCACO)NNH [13,14], HNCO 
[15], CBCACO(CA)HA [16], and HBHA(CBCA)NNH [14]. The ex-
periments, starting with proton magnetization on carbon, were run 
with both short and long refocusing delays to distinguish the carbon's 
protonation state [17]. The spectra were referenced using sodium 2,2-
dimethyl-2-silapentane-5-sulfonate added at the completion of data 
collection, referencing nitrogen and carbon dimensions indirectly 
[18]. Spectra were processed and analyzed using Felix-95 (Molecular 
Simulations). CSI was calculated with the program of Wishart and 
Sykes [19]. Protein modeling was completed using the Quanta pro-
gram (Molecular Simulations) with coordinates obtained from the 
Protein Data Bank. 
3. Results and discussion 
For the sequential assignments of mouse leptin we utilized 
two labeled samples : one with 15N and a second with 15N and 
13C. Incorporation levels were determined to be 98% and 97%, 
respectively. During the analysis of the 15N spectra, we noted 
more signals than expected given the amino acid composition. 
Also a number of NH signals were obviously doubled in a 2:1 
ratio. From numerous experiments we were unable to deter-
mine the source of the heterogeneity and concluded that the 
isomers must be conformational (Kline, unpublished results). 
For this paper only the major isomer will be examined. 
The assignment strategy used is based on identifying an NH 
intraresidual C a , d \ C°, H a and HP signals and then compar-
ing to another NH sequential C a , d5 , C°, H a and HP [20-22]. 
When the intraresidual and sequential signals align, then the 
NHs are defined as sequential. A contiguous series of NHs are 
examined for shift information about their amino acid type. A 
series is subsequently fit into the amino acid sequence. 
Fig. 1 shows a summary of the scalar connections between 
the NHs for leptin which lead to the assignment of the major 
isomer. Gaps occur on this NH-NH connectivity map due to 
the occurrence of proline or missing NH signals (His-26, Gln-
0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved. 
P77S0014-5793(97)00353-0 
240 A.D. Kline et al.lFEBS Letters 407 (1997) 239-242 
VPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILS 
ca 
cß 
C° 
H a 
Hß 
LSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQ 
c« 
cß 
CO 
H« 
Hß 
TSGLQKPESLDGVLEASLYSTEWALSRLQGSLQDILQQLDVSPEC 
c« 
cß 
c° 
H« 
Hß 
Fig. 1. Summation of the sequential NH-NH connectivities which allowed the mouse leptin NMR assignments to be made. A black bar adjoins 
residues when their corresponding NH signals were connected through the indicated intervening atoms. Blanks occur either where no data were 
possible or where no data were observed. 
28, and Gin-100). The connectivity is nearly complete for Ca, 
Ü3, C° and H a which gives confidence in the accuracy of the 
assignments. Fewer residues were connected through the H^, 
due primarily to the limited number of NHs having intrare-
sidual Hß. Despite this, sufficient data were available to 
uniquely assign all the residues. Following the completion of 
the bulk of the assignments, two remaining NHs, Val-1 and 
Thr-27, were assigned principally by elimination. A table of 
the assigned chemical shift values is available upon request to 
the authors. Finally NOE information was used to corrobo-
rate the leptin assignments. 
The CSI is most accurate in determining secondary struc-
ture when a consensus analysis is performed using the four 
nuclei: Ca, C13, C°, and H a [19]. For leptin, these values were 
available for 145 of the 146 residues. The analysis (Fig. 2) 
reveals four long helices and one very short strand segment. 
There are also two relatively long random-coil loops. The 
positions and lengths of the helices match closely with the 
results from the NOE analysis. Occasionally the two methods 
showed slight discrepancies near the end of a helix. For in-
stance, the shifts for Ser-67 indicate a random coil conforma-
tion, while the backbone NOEs are consistent with a-helix. 
These differences never extended more than a single residue. 
Using the CSI and the NOEs, the secondary structure for 
leptin was assigned in the following manner: Helix A runs 
from residues 3-24, Helix B from 51-67, Helix C from 72-
94, and Helix D from 122-141. Each helix contains five to six 
turns. Random coil occupies two long loops 25-50 and 95-
121. The only ß-strand occurs at 47-50, though no NOEs 
were identified linking it to an unidentified strand, so it is 
unlikely that any sheet exists in this protein. The remaining 
loop is a short segment that occurs from 68-71. The length of 
this loop and the presence of proline in the second position, is 
reminiscence of tight turns between ß-strands [23], though this 
is located between helices. While the assignment of secondary 
structure is for the mouse protein, the same assignments are 
likely for the human protein as there are only five amino acid 
changes between the species in the helical regions [1]. 
Leptin's four helices and two interconnecting loops are con-
sistent with a classification as a cytokine four-helix bundle and 
agrees with the earlier threading [11] and CD analysis [12]. 
The characteristic up-up-down-down arrangement of the heli-
ces in the cytokine fold requires long loops between helices A 
and B and between helices C and D [24]. The connector be-
tween helix B and C is generally shorter. The threading anal-
ysis was accurate in its identification of helical residues ; how-
ever, 25 helical residues were missed because they were 
improperly assigned to random coil or ß-strand. The CD es-
timate was close to the 56% measured here, but that study did 
not specify any of the helix positions [12]. 
While in general leptin's secondary structure is similar to 
any four-helix bundle cytokine, in specific, the length and 
position of its helices appears unique. The four-helix motif 
is able to accommodate a wide variation in helix length, so 
A.D. Kline et allFEBS Letters 407 (1997) 239-242 241 
VPIQKVQDDTKTLIKTIVTRINDISHTQSVSAKQRVTGLDFIPGLHPILS 
C S I 
dNN 
dcxN 
d a i N i + 3 
LSKMDQTLAVYQQVLTSLPSQNVLQIANDLENLRDLLHLLAFSKSCSLPQ 
C S I 
dNN 
daN 
d 0 t i N i + 3 
TSGLQKPESLDGVLEASLYSTEWALSRLQGSLQDILQQLDVSPEC 
C S I 
dNN 
daN 
d a ± N i + 3 
Fig. 2. The CSI and NOE information used to determine the secondary structure for mouse leptin. The CSI histogram indicates an a-helix des-
ignation when above the line and a ß-sheet when below. daN indicates the presence of sequential H
a-NH NOE. Thin line designates where day 
was weaker than the intraresidue H°-NH NOE, thick line where daN was stronger than the intraresidue NOE. dNN indicates the presence of se-
quential NH-NH NOE. Thin line designates where dNN was weaker than daN, thick line where dNN was stronger than dK>j. 
Fig. 3. A stereo C a fold drawing of the mouse leptin model, based on the NMR helix assignments. C™ atoms are shown as open circles; termi-
ni are labeled near the bottom of the structure. The 96-146 disulfide is represented by a thin line near the C-terminus. 
242 A.D. Kline et al.lFEBS Letters 407 (1997) 239-242 
that an exact match would be unlikely. Leptin's helix lengths 
are most similar to interleukin-2 [25], interleukin-4 [26] and 
macrophage-colony stimulating factor [27], all members of the 
short-helix family [24,28]. Its single disulfide connection from 
the beginning of the CD loop to the C-terminus, resembles 
granulocyte macrophage-colony stimulating factor [29], an-
other member of the short-helix cytokines. So from secondary 
structure alone, leptin is likely a member of the short-helix 
family. Other parameters important to classification such as 
loop crossover and angles between helical moments [24], will 
have to await the full tertiary structure. 
Computer modeling was undertaken using a superposition 
of the helical bundles for all members of the short-helix fam-
ily. The repetition of hydrophobic residues on leptin's helices 
matched well with the hydrophobic repeats for the known 
structures and was ultimately the basis for aligning the amino 
acid sequences for the modeling procedure. The resulting 
structure for leptin is shown in Fig. 3. The only significance 
we will draw from this model is that the four helices deter-
mined in this study fit nicely into the short-helix cytokine fold 
and have no difficulty making the short turn at 68-71 or the 
disulfide bridge at 96-146. 
The length of its helices, the position of its disulfide, and the 
success of this modeling effort, all suggest that leptin is a 
member of the short-helix cytokine fold. This result is some-
what unexpected due to the receptor's sequence similarity to 
gpl30 [30], potentially connecting it to the IL-6 family of 
receptors [31]. IL-6 [32] and its siblings, leukemia inhibitory 
factor [33] and oncostatin M [34] are all long-helix cytokines 
and have average helix lengths one to two turns longer than 
leptin. Thus while leptin and IL-6 have sequence similarities in 
their receptors, they do not seem to share a ligand structural 
classification. Recent published data also suggests that leptin 
is not a member of the IL-6 family because it does not use the 
signaling protein gpl30 [35]. The leptin receptor is, however, 
not at all similar to the interleukin-2 family of receptors [36], 
thus leptin and its receptor may represent an entirely new 
family of Type I receptors. 
Acknowledgements : The authors gratefully acknowledge Prof. Lewis 
E. Kay (Univ. of Toronto) for providing numerous triple resonance 
pulse sequences and Prof. Brian D. Sykes (Univ. of Alberta) for the 
automated CSI program. 
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Leptin is a four-helix bundle: secondary structure by NMR 

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Leptin is a four-helix bundle: secondary structure by NMR

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