Chapter4

Conformational features of Eledoisin: A non­mammalian NK-2 agonist

4.1. Introduction

Eledoisin, a non-mammalian member of the tachykinin family of neuropeptides, was

first isolated from the posterior salivary glands of two mollusc species Eledone

muschata and Eledone a/dovandi belonging to the octopod Cephalopoda (Erspamer

and Anastasi, 1962). Eledoisin is one of the earliest known members of the tachykinin

family. Isolation of this peptide evoked interest in tachykinins from non-mammalian

sources and has subsequently led to isolation and purification of similar bioactives

peptides from different non-mammalian sources (Severini et al., 2002). Eledoisin is an

undecapeptide with the sequence: pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met­

NH2. Eledoisin exhibits a wide and complex spectrum of pharmacological and

physiological activities, such as powerful vasodilation, hypertensive action and

stimulation of extra-vascular smooth muscle. Extensive studies have been done to

characterize the pharmacological actions of Eledoisin. It was found that Eledoisin

displayed a potent stimulating effect on the mechanical and electrical activity of the

gut (Bertaccini, 1976) and was found to be one of the most active substances on

duodenum (Belloli et al., 1994). Further it was found that Eledoisin has a potent

hypotensive action and intra-cerebroventricular injection of Eledoisin increases plasma

vasopressin in the rat (Massi et al., 1991) with a pronounced increase in vascular

permeability after the injection (Hua et al., 1984). Studies have also shown that

Eledoisin is a very potent anti-dispogenic agent (Tmjan et al., 1990) and it strongly

stimulated amylase secretion from rat parotids (de Caro et al., 1977). Further, studies

have also reported that Eledoisin acts on the hypothalamus to inhibit TRH release, and

its effects are modified by amines of the central nervous system (Mitsuma et al., 1985).

A recent study (El-Agnaf et al., 1998) has shown similar activity and high level of

sequence homology (73%) of Eledoisin with 13 Amyloid protein fragments (Al3 25-35)

and their analogs, which play a major role in the onset and progression of Alzheimer's

disease. Hence there is considerable interest in this peptide as a potential target for

drug design.

76

In comparison to mammalian tachykinins, the affinity of Eledoisin for mammalian

receptors is weak and its selectivity is much less pronounced. It is thought that the E­

type Eledoisin receptor (SP-E) which prefers ligands Eledoisin, Kassinin and NKB

over NKA, Physalaemin and SP, may be a mixture of NK-2 and NK-3 binding sites

(Schwyzer, 1987). A study on tachykinin activity on porcine lower urinary tract

smooth muscle indicated that in addition to NKA, Eledoisin was also potent in

contracting detrusor strips from porcine bladder, which contain mainly NK-2 receptors

(Bolle et al., 2000). Similar results were observed in case of other tissues rich in NK-2

receptors, hence it was concluded that Eledoisin was a NK-2 selective agonist.

Eledoisin being a potent non-mammalian NK-2 selective peptide displaying similar

pharmacological profile as that ofNKA prompted us to investigate the conformational

features of Eledoisin as it may further throw light on structural requirements ofNK-2

receptor binding.

A limited number of studies have been reported on the conformation of Eledoisin (Yu

and Yang, 1992; Wilson et al., 1994). From CD studies Eledoisin has been reported to

assume a beta-structure, resulting perhaps from aggregation, when in contact with

phosphatidyl choline membranes (Schwyzer, 1987) and to form an alpha helical

structure in SDS (Woolley and Deber, 1987). In a study of Eledoisin in DMSO (Yu

and Yang, 1992) by NMR and distance geometry technique, no regular conformations

were found. Wilson and co-workers have reported a detailed study on solution

conformation on Eledoisin, using CD and two-dimensional NMR techniques (Wilson

et al., 1994). In aqueous solution Eledoisin was found to be conformationally

averaged, but assumed alpha-helical structure on addition of 50% TFE or SDS (Wilson

et al., 1994). Their NMR data also indicated that the helical core of Eledoisin was

better defined in SDS micelles environment than in TFE. In TFE/H20 and SDS it was

found that residues 6-1 0 of Eledoisin show more conformational order than the

terminal regions, which undergo dynamic fraying. A possible turn in the N-terminal

"address" region, the putative receptor recognition site of the peptide, was detected by

NMR spectroscopy (Wilson et al., 1994). The NMR data indicated that the helical

central core of Eledoisin was better defined in the micellar environment than in TFE

77

(Wilson et al., 1994). In this study reported here, CD and NMR spectroscopic

techniques have been used to investigate the efeect of different membrane mimetic

solvents on the conformation of Eledoisin.

4.2. Materials and methods

Eledoisin was purchased from Sigma Chemicals (St. Louis, USA). TFE, SDS, Calcium

chloride and DPPG were obtained from Sigma chemicals (St. Louis, USA). DPC was

purchased from Cambridge isotopes (Massachusetts, USA).

4.2.1. Circular dichroism spectropolarimetry

Eledoisin was dissolved in phosphate buffer to make a stock solution of 1mg/250~-tl.

The concentration of Eledoisin in the sample of 300~-tl volume was 116 J..LM. Different

solvents were used to mimic different bio-membrane compartments. The aqueous

environment was mimicked by sodium phosphate buffer at pH 7 .4; TFE mimicked the

hydrophobic interior of the membrane and anionic detergent SDS was used to mimic

the charged surface of the membrane. In our studies TFE solutions were prepared such

that its final concentration in solution after adding peptide would be 1 0, 20, 30, 40, 60

and 90 (% TFE v/v). Similarly SDS titrations of 4, 8, 16, 32 and 64 mM were used in

this study. We have also used lipid micelles and liposomes to study the conformational

behavior of tachykinin peptides. The effect of calcium ions on the structure of

tachykinin peptides was also studied. Calcium chloride was dissolved in TFE to

prepare a stock solution of 2mM. For Ca2+ titrations, aliquots of this stock solution

were added to the peptide solution so as to get the molar ratio of 1:1, 2:1, 5:1, 10:1,

15:1,20:1 and 30:1.

All the CD spectra have been recorded on Jasco J-720 spectropolarimeter (JASCO,

Japan). The spectra have been collected between 190 and 250 nm at room temperature

using a quartz cell having a path length of 1 mm. The spectra are the average over 4

scans each recorded with a bandwidth of 1 nm, 0.25 nm step size and a 0.2 sec time

constant. All the measurements have been performed at room temperature. The spectra

recorded in the presence of DPC, SDS micelles, TFE and liposomes have been

78

corrected by subtracting the spectra of the corresponding DPC, SDS, TFE and

liposome solutions.

4.2.2. NMR spectroscopy

NMR samples were prepared by dissolving 2 mg of Eledoisin in approximately 0.4 ml

of water (90% H20, 10% D20, pH 5.0). The experiments in lipid environment were

performed with an identical peptide sample to which 25 mg of perdeuterated DPC was

added yielding in solution a lipid concentration of 180mM, which is well above the

critical micelle concentration (1mM) for DPC. The lipid to peptide ratio of the NMR

sample was 40: 1. All NMR spectra in DPC and water were recorded on a Bruker DRX

500 (Bruker, Zurich, Switzerland) spectrometer operating at 500 MHz proton

resonance frequency. The data was processed by the XWINNMR program on a Silicon

graphics Indigo workstation (SGI, California, USA).

All the 2D spectra were acquired in the phase-sensitive mode using the TPPI method.

The homonuclear DQF-COSY, NOESY and TOCSY spectra were recorded with 64

scans, a relaxation delay of 1.5 s, a spectral width of 6009.6 Hz in both dimensions,

and with 512 increments in t1 and 2K data points in t2. The TOCSY experiments were

performed using 80ms and MLEV-17 spin lock mixing pulses. The NOESY spectra

were recorded with mixing times of 50, 100, 150, 200 and 250 ms to evaluate the

linear build-up of NOE and to find the mixing time appropriate to the two-spin

approximation. Prior to Fourier transformation the data were multiplied by a 90 °

shifted square sine-bell window function. After zero filling and Sine apodisation in t1

and t2 dimensions, the final size of the data matrix was 1 K x 1 K. All the 2D spectra

were acquired at a temperature of 313 K.

4.2.3. Structure calculation

Distance restraints obtained from NMR data were used to obtain information on the

three-dimensional structure of Eledoisin. For the determination of inter-nuclear

distances, the NOESY peak volumes on the 200ms NOESY spectra were classified as

strong, medium and weak corresponding to the upper-bound inter-proton distance

restraints of 2.7, 3.5 and 5.0 A respectively. Appropriate pseudoatom corrections were

79

applied to non-stereo specifically assigned methylene and methyl protons. A total of

166 NOE constraints (68 intra residue constraints, 55 constraints of i to i+ 1, 11

constraints ofi to i+2 and 28 constraints ofi to i+3) were applied for Eledoisin. A total

of 50 structures were initially generated for both the peptides using DYANA (Giintert

et al., 1997). Dihedral angles (<l>), which were derived from the measured 3JNH values,

were also used as constraints. The 20 conformers with the lowest target function

values (i.e. least violations of experimental restraints and Van der Waals distances)

were subsequently subjected to restrained energy minimization.

4.3. Results

4.3.1. CD studies on Eledoisin

The effect of different solvents on the conformation of the nonmammalian NK-2

selective agonist Eledoisin was studied using CD spectropolarimetry. The spectrum in

aqueous solution shows the peptide being primarily unstructured, while addition of the

structure inducing TFE, SDS and DPC micelles induced a shift towards helical

conformation (at concentration as low as respective CMC) (Figure 4.1 ). Anionic

DPPG liposomes were also found to induce a helical structure in Eledoisin (Figure

4.5). The titrations were performed with various concentrations of TFE (Figure 4.2)

and SDS (Figure 4.3) micelles. The CD results indicate that Eledoisin interacts with

TFE and SDS micelles undergoing a conformational transition between a prevalently

random coil state (in water) to a-helical state as indicated by an isodichroic point at

203nm in both the titrations. The observation of an isodichroic point indicates a two­

state equilibrium, which can be described by a single equilibrium constant. The helix­

coil transition curve further supports this (Figure 4.4). R1 and R2 values calculated for

Eledoisin in buffer, TFE, DPC, SDS and DPPG (Table 4.1) show an induction of the

helical structure on addition of TFE, SDS, DPC and DPPG liposomes.

Studies have shown that contractile action of NK-2 agonists are dependent on

extracellular calcium (Riordan et al., 2001; Matran et al., 1988), hence it was of

interest to study the effect of calcium ions on conformation of Eledoisin. Our CD

calcium titration studies on Eledoisin (Figure 4.6) did not show any significant change

80

..:;--8 (.)

0£ ~

'"0 '-' ,-.. CD '-'

a ·u ;g ~ ..... ro 0 20L---~-----L-----L ____ J_ ____ ~----L-~~ a 19o zoo 220 240 2eo

Wavelength(nm)

Figure 4.1: CD spectra of Eledoisin in buffer (solid line), 60%TFE

(dashed line) and 16mM SDS (dotted line) and DPC

(dash-dot line)

0 E

"' ........ E ()

Ol Q)

~

.....

3e+5.--------------------------------------------------.

2e+5

1e+5

ro -1e+5 0 E

-2e+5+--------.-------,--------,--------,-------.------~

190 200 210

- · · wavelength vs 1 O%tfe -- waveleng1h vs 20%tfe · · · · · wavelength vs 30%tfe -- wavelength vs 40%tfe --- wavelength vs 60%tfe -- wavelength vs 90%tfe

220 230 240

wavelength (nm)

Figure 4. 2: CD spectra ofTFE titrations of Eledoisin

1.2e+S

0 1.0e+S

N~ 8.0e+4

(J

Cl 6.0e+4 Q)

~ 4.0e+4

~ :?:;- 2.0e+4 ·u g. 0.0 Q) ..... -2.0e+4 cu 0 E -4.0e+4

-6.0e+4

190 200 210

- · wavelength vs sds4 · · · wavelength vs Ssds

- · · wavelength vs 32sds -- wavelength vs 16sds

220

wavelength (nm)

230 240

250

250

Figure 4. 3: CD spectra of Eledoisin in presence of different concentrations

ofSDS

0

-2e+4

E -4e+4 c N N N

ro §: -6e+4

-8e+4

-1e+5

0 20 40 60 80 100

% of TFE (v/v)

Figure 4. 4: Helix -coil transition curve of Eledoisin in TFE

z-"(3 0

g a; iii -10000 0 E

-20000 +-----,-----,------~----.------r----1 190 200 210 220 230 240 250

Wavelength (nm)

1- wavelength vs dppg

Figure 4. 5: CD spectra of Eledoisin in presence ofDPPG liposomes.

Table 4. 1: Analysis of helical content in Eledoisin in presence of different solvents

using various parameters (as listed in Chapter 2)

Solvent Rt R2 % Helicity Mean helical content

Buffer 0.35 0.15 24 0.28

TFE -1.8 0.73 94 0.95

SDS -1.3 0.8 86 0.91

DPC -1.2 0.5 74 0.78

DPPG -1.7 0.57 36 0.38

-0 E

N"-E (.)

C> Q) u -CD' ->. +-' "() :;:::> Q.

Q) ...... co 0 E

4e+5

3e+5

2e+5

1e+5

0

-1e+5

-2e+5

190 200 210

- · · wavelength vs 100%tfe -- wavelength vs ca 1:1 - · · wavelength vs ca 2:1 · · · · wavelength vs ca 5:1 -- wavelength vs ca 10:1 -- wavelength vs ca 15:1

wavelength vs ca 20: 1 - · wavelength vs ca 30: 1

220 230 240

wavelength (nm)

Figure 4. 6: CD spectra of calcium titration of Eledoisin

250

in conformation. A slight increase in helicity at low concentrations of Calcium was

observed and a subsequent decrease in helicity was observed at higher concentrations

of Calcium. However the magnitude of the effect was considerably weak and does not

exhibit a significant change in conformation as observed for NKA.

4.3.2. NMR studies on Eledoisin

Some preliminary 1D and 2D spectra of Eledoisin were recorded in aqueous solution

at various temperatures. NMR spectra in water indicated presence of aggregation. No

further study was carried out in water because of peptide aggregation and also since

CD results indicated random structure of Eledoisin in water. Aliquots of d3s-DPC were

then added to an aqueous solution of Eledoisin and a series of 1D and 2D proton NMR

spectra were recorded at 500 MHz. The structural stabilization was apparent in the

NMR spectra on addition of 12 mg or more of DPC. All subsequent experiments were

performed under these solution conditions (DPC concentration 180mM; lipid to

peptide ratio 40: 1 ). Some of the residues of Eledoisin have a minor conformer in DPC.

The peaks from the minor conformer could not be suppressed by temperature or by pH

variations. Since all the resonances of the minor conformer could not be seen clearly in

the spectrum, the structure of the minor conformer could not be obtained.

4.3.2.1. Spectral assignment

Assignment of the proton spectra of Eledoisin in the presence of membrane mimetic

solvent (DPC) was accomplished using the technique of sequence-specific resonance

assignments developed by WUthrich (WUthrich, 1986). The spin systems of individual

amino acids were identified from TOCSY (Figure 4.7) and DQF-COSY (Figure 4.8)

experiments. The assignment was straightforward and unambiguous as each of the

amino acid within the sequence of Eledoisin is unique and non-degenerate. Sequential

connectivity was identified from the NOESY spectra. A cross peak between the amide

proton of Ser 3 and the a-H proton of Pro 2 in the NOESY spectra confirmed the

assignment of Pro 2 spin system. The assignment of the various resonances in the

sequence of the peptide is indicated on the spectrum (Figure 4.9a). The amide region

of the NOESY (200 ms mixing time) spectrum of Eledoisin is shown in Figure 4.9b.

81

K4 18 A6

Mll

LlO

D5

F7

09

6.6 8.4 8.2 8,0 1.8 ppm

Figure 4. 7: Fingerprint region of the 500 MHz TOCSY spectra of Eledoisin in

DPC micelles

C& f•

J

1;!.4.

G9

oO ~·

A6

... I I

li.:Z

JS

1- 4.0

n t I

7.lil

Figure 4.8: Fingerprint region of the 500 MHz DQF-COSY spectra of

Eledoisin in DPC micelles

ppm

(a) 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4;5

ppm

(b)

8.0

8.2

8.4

8.6

05 G9

K4

1l

GD

18 L10 F7 M11

,.,~ea G9/M11

fl. ~5JF7 K4/A6 •0 "o I

"'"""I'"""'""" "I"""""""' I"""'""" '"I""'"""'" "I"'"'''"" "I'"""'""" "I"""'""""' I""""""' "I""'""

8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 ppm

Figure 4. 9: (a) The NH-a,~,y region of the 500 MHz NOESY spectrum of

Eledoisin in the presence of membrane mimetic solvent (DPC), recorded with a

mixing time of 200 ms. (b) The NH-NH region of the 500 MHz phase-sensitive

NOESY spectrum of Eledoisin in the presence of membrane mimetic solvent

(DPC), recorded with a mixing time of200 ms.

Table 4. 2: Proton NMR assignments of Eledoisin in the presence of membrane

mimetic solvent (DPC)

Residue NH aH J3H Others

PGlu 4.31 2.68, 2.52 yCHz: 2.43, 2.06

Pro 4.55 2.39 yCH2: 2.13, 2.06

8CH2 : 3.85, 3.73

Ser 8.25 4.53 4.08, 3.97

Lys 8.57 4.27 1.95, 1.93 yCH2 : 1.56

8CHz: 1.79

ECH2 : 3.06

ENH3: 8.79

Asp 8.32 4.60 2.86, 2.79

Ala 8.13 4.25 1.41

Phe 8.04 4.51 3.28, 3.21 Aro: 7.32

lie 7.95 3.98 2.01 yCHz: 1.66

yCH3: 1.33

8CH3: 0.98, 1.01

Gly 8.19 3.98

Leu 7.84 4.33 1.85 yCH3: 1.69

8CH3: 0.98, 1.01

Met 7.88 4.43 2.19 yCH2 : 2.54, 2.65

ECH3: 2.11

NHz 7.31, 7.15

Complete proton resonance assignments for Eledoisin in the presence of membrane

mimetic solvent (DPC) is summarized in Table 4.2.

4.3.2.2. Analysis of chemical shift values

The a-H proton chemical shifts for Eledoisin in DPC were analyzed and the results

show that for residues 4-8 the difference in the chemical shifts are continually

negative, which suggests that in this part of the peptide a helical secondary structure is

favored in the presence of DPC (Figure 4.1 0).

It is very difficult to determine the exact on/off rates for the binding of Eledoisin with

DPC micelles from the NMR data. However the percentage of bound conformers to

the free conformer may be obtained by determination of the amount of a-helix

observed (Rizo et al., 1993). The semi quantitative estimate of helical content for

residues 4 through 11 for Eledoisin in DPC micelles thus calculated was 31.5%. The

prediction of helical content for Eledoisin (33%) using the prediction algorithm

AGADIR (Munoz and Serrano, 1994) correlates well with the results from the NMR

data.

4.3.2.3. Analysis of NOE connectivities

A summary of sequential and medium range NOE connectivities observed for

Eledoisin in the presence of DPC micelles is given in Figure 4.11. For Eledoisin (Ser

3-Met 11), intra-residue cross peaks are more intense, which indicates the presence of

helical structure. Similarly, variations in relative intensities of dNN and duN sequential

NOEs support the proposal of a helical structure for Eledoisin (Ser 3-Met 11) as dNN

contacts are stronger than daN contacts (Wuthrich et al., 1984).

A dense grouping of NOEs (Figure 4.11), the six duN (i, i+3), five du13 (i, i+3), eight

sequential dNN NOEs and three dNN (i, i+2) NOEs coupled with three duN (i, i+2) NOEs

support the presence ofhelical structure in this region of Eledoisin (involving residues

3-11 ). The presence of some population of a-helix is supported by the observation of a

daN (i, i+4) connectivity between Asp 5 and Gly 9. However, the observation of four

82

0.15 .--------------------------,

0.1

E 0.05

&: 0 +-"....____~ r:: ~ -0.05 ..: ~ -0.1 Ui' .c -0.15 0

c:o -0.2

-0.25 -0.3 __L,__ ____________________ __j

2 3 4 5 6 7 8 9 10 11

residue number

Figure 4. 10: a-H proton chemical shifts for Eledoisin in presence of DPC

micelles

d NN(I, 1+1)

ctxN (1, 1+1)

df3N (I, 1+1)

dNN(I, 1+2)

daN (I, 1+2)

4tN (I, 1+3)

q:xf~(l, 1+3}

.... (1, 1+4) ""r.tN

BPSKDAF G L M

-

Figure 4.11: The NOEs that are important to characterize the secondary

structure of Eledoisin in the presence of DPC micelles. Filled triangles refer to

a coupling constant of 4 to 5 Hz and filled circles to 6 Hz and above.

daN (i, i+2) connectivity between Ser 3-Asp 5, Asp 5-Phe 7, Ala 6-Ile 8 and Phe 7-Gly

9 is more consistent with a 310 helix than a a-helix in this region. Further, daN (i, i+2)

crosspeaks observed for the C-terminal residues are stronger than the daN (i, i+ 3)

counterparts, indicating that the folded C-terminus is 310 helix like in character.

Overlap of some of the resonances under water interfered with the NOE analyses.

However, the number of aHi - J3Hi+3 NOEs observed indicates that Eledoisin is

substantially folded from residues 3-11. Observation of these various types of NOEs

simultaneously suggests that some degree of conformational averaging is present

around a predominantly helical core. In theN-terminus of the helical segment there are

cross peaks characteristic of a J3-turn or 3w-helix. Therefore, the possibility of turn

conformations must be considered for the region near the N-terminus as seen by the

presence of daN (i, i+2) NOE between Ser 3-Asp 5 and Asp 5-Phe 7. In these

structures, distances daN (i, i+2) and cia~ (i, i+2) are very short and the NOE cross

peaks are usually observed (Wuthrich et al., 1984; Dyson et al., 1988). Moreover, side

chains of polar residues such as Asp and Ser when preceding helices, can provide

stabilizing C=O donors for hydrogen bond interactions in helices ... This type of

interactions is especially important for stabilizing short length helix in a small linear

peptide.

All measurable coupling constants for Eledoisin are in the range of 4-6 Hz, with the

exception of Ser 3 (6.4 Hz) and Met 11 (7Hz). The apparent 31HNa coupling constant

is a weighted average of the population and depends on the distribution of angles over

the population (Kessler et al., 1988). Thus the low31HNa values (< 6 Hz) suggest that

there is a large population of helical structures in the stretch between residues 4-11.

Met 11 also shows a large 3JHNa (>6Hz), which may be due to fraying of helix at the

terminus.

The temperature dependency of amide proton chemical shifts is a simple probe that

provides information on the involvement in hydrogen bond formation or sequestering

from the solvent. A low dependency of the amide chemical shift on temperature in

aqueous environment, i.e., temperature coefficient > -5 ppb/K, is usually indicative of

83

Table 4. 3: Temperature coefficients for Eledoisin in presence of DPC

Residue Temperature coefficient (!!.51!!. T) in ppb/K

Ser 3 -4.7

Lys 4 -7.8

Asp 5 -4.9

Ala6 -5.1

Phe7 -3.4

Ile 8 -5.4

Gly9 -2.9

Leu 10 -2.7

Met 11 -1.7

the presence of a stable hydrogen bond (Dyson et al., 1988; Anderson et al., 1992 &

1997). For solvent-accessible amide protons, which are more sensitive to a temperature

change, the slope is generally more negative than -5 ppb/K. Several 1D spectra for

Eledoisin were collected under different temperatures ranging from 290 K to 320 K.

The temperature coefficient values for the amide protons of Eledoisin are shown in

Table 4.3. On the basis of the above criteria, the stable amide protons of Eledoisin with

low solvent accessibility via hydrogen bond formation were identified. Phe 7, Gly 9

and Leu 10 shows temperature coefficient values considerably more positive than -5

ppb/K indicating that these residues might be involved in hydrogen bonding.

From the above NMR results, it is concluded that the residues 3-11 clearly meet the

criteria for the existence of helical structure: presence of sequential dNN (i, i+ 1) cross

peaks, presence of medium range daN (i, i+ 3) and daJ} (i, i+ 3) cross peaks and a series

of 3JHNa coupling constants of 6Hz or less. Further, the temperature coefficients of

several residues in this region are indicative of presence of hydrogen bonds thereby

confirming the presence of a folded conformation in this region. However, the entire

peptide is not helical. Due to the lack of medium range cross peaks and the large 31HNa

coupling constants, we conclude that the first three residues are in tum conformation.

No evidence for helix stabilization through salt bridge formation was observed.

4.3.3. Generation of three-dimensional strcuture

It was of interest to use the observed NOEs to obtain information on the three­

dimensional structure of the peptide (Table 4.4), as NOE data indicated the presence of

a helical structure along the central core of Eledoisin in DPC. This was done using the

torsion angle dynamics algorithm for NMR applications, DYANA (Guntert et al.,

1997). Initially, 50 structures were generated by DYANA using simulated annealing

protocol, which improves the convergence of the structure calculations by introducing

redundant dihedral angle restraints. The 20 conformations with the lowest target

function value (i.e. least violations of experimental restraints and Van der Waals

distances) were chosen for further refinement using restrained energy minimization

(Table 4.5). The resulting structures are shown in Figure 4.12, after superimposing the

84

Table 4. 4: The upper distance restraints used in DYANA to calculate 3D structure of Eledoisin

1PGLU HA 1 PGLUHB2 4.00 8ILE HA 1 PGLUHB3 4.00 HN 5 ASP HA 3.40

2PRO HN 7PHE HA 3.50 HD2 1PGLU QB 4.50 HN 6ALA HA 3.80 HD2 1PGLU HG2 4.50 HN 8 ILE HA 3.00 HD3 1PGLU QB 4.50 HN 7PHE HB2 3.50

3 SER HN 7PHE HB3 3.50 HN 4LYS HN 2.80 HN 8 ILE QG2 4.50 HN 2PRO HA 3.50 HN 8 ILE QD1 4.00 HN 3 SER HA 3.00 HA 11 MET HB2 4.40 HN 2PRO HD2 2.80 HA 11 MET HB3 4.40 HN 2PRO HD3 3.50 HA 8 ILE HB 3.50 HN 2PRO QB 4.00 HA 8 ILE QG2 3.50 HA 3 SER HB2 3.50 HA 8 ILE QG2 3.50 HA 3 SER HB3 3.50 HA 8 ILE QD1 4.50

HA 6ALA QB 4.50 QG2 8 ILE HG21 3.50 QG2 8 ILE QD1 4.50

4LYS 9GLY HN 5 ASP HN 2.80 HN 8 ILE HN 2.80 HN 6ALA HN 4.20 HN 10LEU HN 2.80 HN 4LYS HA 3.00 HN 11 MET HN 4.20 HN 3 SER HA 3.50 HN 6ALA HA 3.40 HA 7PHE HB2 4.40 HN 9GLY QA 3.00 HA 7PHE HB3 4.40 HN 8 ILE HA 3.50 HN 3 SER HB2 4.00 HN 8 ILE HB 3.50 HN 3 SER HB3 4.00 HN 5ASP HA 5.00

5 ASP 10LEU HN 6ALA HN 2.80 HN 7PHE HA 3.40 HN 7PHE HN 4.20 HN IOLEU HA 3.00

HN 5 ASP HA 3.00 HN 9GLY QA 3.40

HN 4LYS HA 3.50 HN IOLEU QB 3.50

HN 5 ASP HB2 4.50 HN 10LEU HG 4.50

HN 5 ASP HB3 4.50 HN IOLEU QQD 4.50

HA 8 ILE HB 3.50 HA IOLEU QB 3.50

HN 4LYS QB 4.00 HA 10LEU HG 4.50

HN 3 SER HA 3.80 HA IOLEU QQD 4.50

6ALA QB 10 LEU HG 3.50

HN 7PHE HN 2.80 QB 10 LEU QQD 3.50

HN 5ASP HA 3.50 11 MET HN 3 SER HA 3.40 HN 10 LEU HN 2.80

HN 6ALA HA 3.00 HN II MET HA 3.00

HN 6ALA QB 3.50 HN 10 LEU HA 3.50

HN 5 ASP HB2 3.50 HN 8 ILE HA 3.40

HN 5 ASP HB3 3.50 HN 11 MET QG 4.50

7PHE HN II MET QB 3.50

HN 8 ILE HN 2.80 HN 10LEU HG 4.00

HN 5 ASP HA 3.80 HN 10LEU QD2 5.00

HN 4LYS HA 3.40 HA 11 MET HG2 3.50

HN 6ALA HA 3.50 HA 11 MET QG 4.50

HN 7PHE HA 3.00 HA 11 MET QB 4.50

HN 7PHE HB2 3.50 HA 11 MET QE 4.50

HN 7PHE HB3 3.50 HN 10LEU QB 3.50

HN 6ALA QB 3.50 HA 7PHE HB2 3.50 HA 7PHE HB3 3.50 HA 10 LEU QB 4.40 HA 9GLY HN 3.80 HN 6ALA QB 4.40

Table 4. 5: Structural statistics for twenty structures calculated for Eledoisin using

DYANA

str target upper limits lower limits van der Waals torsion angles

function # sum max # sum max # sum max # sum max

1 7.64E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 O.Ql

2 8.37E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 O.Ql

3 8.37E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

4 8.37E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

5 8.37E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

6 8.37E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

7 8.38E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

8 8.38E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

9 8.43E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

10 8.44E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

11 8.51E-02 0 0.9 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

12 0.13 0 1.1 0.18 0 0.0 0.00 0 0.2 0.06 0 0.0 0.01

13 0.14 0 1.2 0.19 0 0.0 0.00 0 0.2 0.06 0 0.0 0.02

14 0.19 0 1.5 0.16 0 0.1 0.05 0 0.2 0.06 0 0.0 O.Ql

15 0.24 0 1.7 0.18 0 0.1 0.05 0 0.3 0.06 0 0.0 0.01

16 0.95 0 0.8 0.16 0 0.0 0.00 4 1.6 0.55 0 0.0 0.01

17 0.95 0 0.8 0.16 0 0.0 0.00 4 1.6 0.55 0 0.0 0.0 I

18 0.95 0 0.9 0.16 0 0.0 0.00 4 1.6 0.55 0 0.0 0.01

19 1.01 0 1.1 0.19 0 0.0 0.00 4 1.7 0.55 0 0.0 0.02

20 1.05 0 1.4 0.16 0 0.1 0.05 4 1.7 0.55 0 0.0 0.01

Ave 0.33 0 1.0 0.16 0 0.0 O.Ql 0.5 0.18 0 0.0 0.0 I

+/- 0.38 0 0.3 0.01 0 0.0 0.02 2 0.7 0.21 0 0.0 0.00

Min 7.64E-02 0 0.8 0.16 0 0.0 0.00 0 0.1 0.06 0 0.0 0.01

Max 1.05 0 1.7 0.19 0 0.1 0.05 4 1.7 0.55 0 0.0 0.02

Constraints violated in 6 or more structures:

# mean max. I 5 10 15 20

0 violated distance constraints.

0 violated angle constraints.

Pairwise RMSDs for residues 1 .. 11:

Mean global backbone RMSD: 0.39 +/- 0.21 A (0.04 .. 0.83 A)

Mean global heavy atom RMSD: 1.31 +/- 0.36 A (0.56 .. 2.20 A)

C-terminus

AS

S3

N-terminus

Figure 4. 12: Stereo view showing the superimposition of the backbone atoms

of eledoisin for 20 structures generated by DYANA.

ISII

111 II

-'Ill

-I Sll -+-----"T"""-....l-...:..-r-__..;.---.-----....:....j -I Sll -')() II lj() lSI I

ll

Figure 4.13: Ramachandran plot of all 20 refined structures of Eledoisin.

backbone atoms. Pair wise RMSD calculated for backbone atoms for residues 1 to 11

for all 20 refined structures ranged from 0.04 - 0.83 A, with a mean value of 0.39 A

and a standard deviation of 0.21 A. The ensemble of strutures has been deposited in

Protein Data Bank (PDB ID code: 1MXQ).

In a stable secondary structure, both <I> and \f' dihedral angles should have well-defined

values. The Ramachandran plots of all 20 refined structures (Figure 4.13) indicate that

the backbone dihedral angles consistently lie in the a-region and are solely within the

allowed ranges. A helical-type backbone arrangement is indicated for the central­

region of Eledoisin, in particular the stretch from Ser 3 through Met 11 (Figure 4.12),

with some dynamic fraying of the helix termini. Measurement of C= Oi, NHi+3 versus

C=Oi, NHi+4 distances along the stretch was made and i, i+ 3 distances correlated with

the Eledoisin having a preference for 310 helix over regular helix. For theN-terminus

of Eledoisin the aHi-aHi+3 distances were measured to be within 7 A in ensemble of

conformations obtained. This distance is the threshold for defining a 13-tum. However

it may not be appropriate to interpret this data in terms of a single tum conformation

and the more likely situation is that Eledoisin exists in equilibrium between two states,

one in which there is a tum in the N-terminus followed by a 310 helix and another

wherein the helix extends from Lys 4 to Met 11.

4.4. Discussion

Our CD studies on Eledoisin show that the peptide is primarily unstructured in an

aqueous solution, while in presence of polar solvents and membrane mimetic micelles

like SDS and DPC a helical conformation is induced in this peptide. Though Eledoisin

is a neutral peptide with a net charge of zero, it was found to interact well with anionic

micelles and liposomes. It was observed from our CD data that a helical structure was

induced for Eledoisin even at SDS concentrations well below the erne. Our studies on

effect of Calcium ions on the conformation of Eledoisin did not show any significant

change in conformation as compared to NKA and the spectra displayed only slight

increase in helicity with increase in calcium concentration.

85

NMR studies reported here indicate that in aqueous solution Eledoisin prefer to be in

an extended chain conformation whereas in the presence of membrane mimetic

solutions helical conformation is induced. The NMR studies reported here suggest that,

in hydrophobic environment, part of the address domain and the whole of the message

domain are folded. In presence of DPC micelles Eledoisin adopts a helical structure

wherein the helical region extends over residues from 3 through 11. This observation

is supported by structure activity data reported by Cascieri and coworkers (Cascieri et

al., 1986), suggesting that Eledoisin binding (NK-2 I NK-3) site has a requirement for

a folded conformation of the C-terminal pentapeptide. The likely situation is that

Eledoisin exists in equilibrium between two states in which there is a possibility of a

tum over residues 1-3 followed by a stretch of helical structures for residues 4 through

11, another where the helical region extends over residues from 3 through 11.

Extension of the helix in Eledoisin towards the N-terminus past Asp 5, which is

considered to be a helix breaking residue is consistent with the report that classical

helix-breaking residues can often be accommodated in helices in membrane -mimetic

environments (Li and Deber, 1993). In DPC, the structural equilibrium is biased

towards a 310-helix from residues 3 through 11, though small populations of regular a­

helix cannot be excluded in the solution ensemble since 3w-helices are intermediates

in the folding/unfolding pathways of regular helices. Also short linear peptides like

Eledoisin may be too short to sustain a well-defined regular helix in solution. The

amidated C-terminus of Eledoisin in DPC comprises 3w-helix with some possible

fraying of helix terminus and such a dynamic fraying of helical terminus is expected

for small linear peptides.

The "address" segment of Eledoisin, while undergoing greater conformational

averaging than the message domain, also retains substantial conformational order in

DPC. This order may be interpreted as a loosely defined tum or an unstable

continuation of 310-helix along the message domain. However, the stability of turns is

not known since there are few reports on turns occurring in membrane mimetic

solvents (Sonnichsen et al., 1992). However, identification of folded conformation in

N-terminus under hydrophobic conditions has some significance, as it may represent

86

an essential feature ofNK-2 I NK-3 binding. It is significant to note that N-terminus of

NK-2 selective agonists, NKA and Neuropeptide K were also found to be folded in

TFE (as reported in Chapter 3; Chandrashekar and Cowsik, 2003; Horne et al., 1993).

The results from our structural studies agree well with structure activity data available

for Eledoisin (Casiceri et al., 1992; Badgery-Parker et al., 1993; Buck and Shatzer,

1988; Severini et al., 2000). It can be seen from Figure 4.14 that the C-terminus

presents a hydrophobic upper half comprising of the Phe 7, Ile 8, Leu 10 and Met 11

residues and theN terminus a hydrophilic lower half comprising of the Ser 3, Lys 4

and Asp 5 residues. We postulate that the hydrophobic C-terminus interacts with the

transmembrane region of the receptor with Phe 7, Leu 10 and Met 11 forming the

anchoring points and contributing a major portion of the binding energy. The

hydrophilic and solvent accessible N-terminus helps to maintain the peptide

conformation and plays an important role in NK-2 receptor binding with Lys 4 and

Asp 5 as anchoring points. It is interesting to note in this context that the modification

of residues Lys 4, Asp 5 of Eledoisin as reported in binding assays (Casiceri et al.,

1992; Buck and Shatzer, 1988) modulates its affinity and selectivity for the receptors.

A recent study on analysis of the role of the amino terminal domain of tachykinins in

NK-1 receptor signaling and desensitization, indicated that NKA, Eledoisin and

Kassinin were full agonists of NK-1 but is able to cause only partial homologous

desensitization of the receptor as compared to SP (Vigna, 2003). This has been

attributed to the differences in the structural elements in the N-terminal domain of

these peptides in comparison to SP. It is interesting to note in this context that NKA

and Eledoisin adopt similar structures in their N-terminus and overall structural

homology between the two peptides is high. These conformational features may be

important in determining their NK-1 receptor desensitizing activities.

In conclusion the results obtained in this investigation are consistent with the proposed

biologically active conformation ofNK-2 receptor agonist. Moreover, the NMR results

presented here agree well with the theoretical secondary structure prediction for turns

and helices in Eledoisin using program ALB by Wilson and coworkers (Wilson et al.,

87

Figure 4.14: A graphic representation of the lipid-bound Eledoisin

conformation. The peptide backbone is shown as a ribbon tube (blue). [onic

residues are colored red, polar residues are colored purple and the hydrophobic

residues are colored yellow. The helical segment is clearly visible.

1994 ). Our studies indicate that the helical central core of Eledoisin is better defined in

DPC micelles than in SDS and TFE. An increase in helical content is observed in

presence of lipid micelles with the helix extending from residue 3-11, in comparison to

SDS wherein the helix extends from 6-11 (Wilson et al., 1994). The presence of a

loosely defined turn in the N-terminus preceding the helical core in the C-terminus of

Eledoisin is consistent with that observed in SDS and TFE. The overall conformational

features adopted by Eledoisin in DPC micelles correlate well with that reported for

Eledoisin in TFE and SDS micelles (Wilson et al., 1994) and with those ofKassinin in

DPC (Grace et al., 2001). In bioassays, Kassinin has been found to interact withE-type

Eledoisin receptors in much the same manner as Eledoisin (Erspamer et al., 1980;

Iversen, 1982). On the basis of this correlation, it is interesting to note that

conformation adopted by Eledoisin in the presence of DPC micelles provides a

biologically relevant structure.

88