Conformational analysis of human growth hormone [6-13] peptide analogues

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<ul><li><p>Int. J. Prpridr Protein R ~ J . 48, I Y Y 6 . 1- I 1 Printed in UK all ri,qhts rcsrrrrd </p><p>PEPTIDE &amp; PROTEIN RESEARCH ISSN 0367PXj77 </p><p>Conformational analysis of human growth hormone [ 6-13] peptide analogues </p><p>KERRY A. HltiGINS. PHILIP E. THOMPSON and MILTON T.W. HEARN </p><p>Centre for Bioprocess Technology. Deptrrtineiit of' Biocheinistrj. anti Mo~eciilur Biolog?,, Monash University, Clayton, C'ictoriu, Aiisfrulici </p><p>Received 28 March, revised 30 August. accepted for publication 22 October 1995 </p><p>The conformational analysis of a series of ten hGH[6-131 peptide analogues is reported. As part of our earlier studies, the r-aminosuccinimide modified fragment Asu1'-hGH[6-l 31 has previously been identified as a potentiator of insulin activity in intravenous insulin tolerance tests, and various analogues have been subsequently designed, synthesised and employed to acquire structure-activity data. These studies have lead to the conclusion that the conformational characteristics a t the C-terminus of each of the active peptide analogues is important to the biological activity. In the present investigation, molecular dynamics and simulated annealing techniques have been used to examine the accessible conformational states of the C-terminal region of ten different hGH[6-13] peptide analogues. Of these six are active peptide analogues while the other four show no biological activity. Examination of the conformer groups identified using this molecular dynamics approach showed a common conformational motif for each of the active peptides. Q Munksgaard 1996. </p><p>Key words; computer modelling: molecular dynamics: peptide analogue: peptide conformation; simulated annealing </p><p>Discrete fragments of human growth hormone (hGH) show insulin-like activity. A number of previ- ous investigations have concluded that the N-terminal peptide region of hGH was responsible for this activ- ity ( 1-4). The r-aminosuccinimide modified fragment Asu"-hGH[6-13] (1) was subsequently identified as an in vivo potentiator of insulin activity with intraven- ous insulin tolerance tests and a modulator of glyco- gen synthesis in other in vitro bioassays ( 5 , 6). The observed activity makes this octapeptide and particu- larly the derived ~-lactam1'-hGH[6-l 31 analogues attractive lead compounds for the development of new therapeutic agents for potential use in type I1 diabetes treatments. </p><p>Previous investigations in this laboratory have sought to identify the mechanism by which this octapeptide and its analogues exert their biological effects. Various analogues of the a-aminosuccinimido- peptide (1) have been designed, synthesised and subsequently used to accumulate detailed structure- activity data (1-4). It has been found that within the N-terminal region of these peptide analogues the chemical composition of the amino acid side chains represents the dominant determinant of activity. In contrast, these studies have shown that it is the conformational characteristics of the C-terminal </p><p>region of these peptide analogues rather than their chemical compositional features per se that appear to be far more important for the expression of the biological activity by this sequence region. In particu- lar. substitution of the metabolically unstable Am"- Asn" segment by a range of other structural entities with similar conformational features has lead to the development of peptide analogues with similar or enhanced potencies in the intravenous insulin toler- ance test (IVITT) in the both the normal and the diabetic rodent animal models (6-1 1). Figure 1 illus- trates examples of these active peptide analogues. It has previously been suggested that a Type 11' p-turn conformation may occur in this Asu"-AsnL2 region of the hGH[6-131 derived peptide (1). In order for our analogue design programme to develop further, it was important to examine in detail the nature of the conformational constraints imposed by the amino acid residues at positions Aaa( 11) and Aaa( 12) in peptide (1) and its active variants. </p><p>Molecular dynamic/simulated annealing techniques are increasingly being applied as an integral compon- ent of the rational design of peptide analogues and peptidomimetics (12, 13). The ruison d'ttre of many previous molecular dynamics/simulated annealing studies with peptides and proteins has been the ulti- </p><p>1 </p></li><li><p>K.A. Higgins et al. </p><p>Peptide (7): Pepode (8): hGH 16-131: </p><p>Leu-Ser-ArgLeu-Phe- X- Y- AlaNH2 </p><p>Peptide (1): Pepbde (2): </p><p>Pepllde (3) Peptide (4) </p><p>Peptide (6): Peptide (5): </p><p>FIGURE 1 The amino acid sequence of the insulin potentiating peptide, hGH(6-13), and each of the active XY peptide fragments where peptide 1 incorporates the Am"-Am", peptide 2 the 3-amino-y- lactam"-Gly", peptide 3 the 4-arnino-y-la~tam"-Gly'~, peptide 4 the ~-Ala"-Pro'', peptide 5 the D-Pro"-Asn'' and peptide 6 the /l-Asp"-Asn" residues. The d2, $', d3 and $3 dihedral angles for each of the peptides are illustrated. </p><p>mate aim to identify the conformation at the global minimium. Our aim in this study was to identify the conformations available to each of the peptide frag- ments. In high-temperature molecular dynamics simu- lations, the available thermal energy is used to climb and cross-conformational energy barriers. Consequently, molecular dynamics (MD) provides information into accessible conformational states of the molecule. Previously M D procedures have been used with a variety of small peptide systems such as vasopressin ( 14) and gonadotropin-releasing hor- mone (14, 15) to find low-energy conformational states across energy barriers that would be inaccess- ible to classical minimisation strategies. In the investi- gations described in this paper, a similar molecular dynamics/simulated annealing approach has been used to examine the conformations available to a series of ten peptides of the general sequence Leu- Ser-Arg-Leu-Phe-X-Y-AlaNH, in which the X-Y res- idues have been substituted. Both active (Fig. 1) and the inactive (Fig. 2) peptides were investigated. Our primary interest in this study was to examine the conformations available to the C-terminal region of the peptide, and in particular, the X-Y fragment. </p><p>2 </p><p>Peptlde (9) Peptlde (10) </p><p>FIGURE 2 The XY sequence for each of the inactive peptides investigated. Peptide 7 incorporates the L-Pro"-GlylZ, peptide 8 the ~-Asp"- Am", peptide 9 the L-Aka"-Pro" and peptide 10 the /&amp;Asp"- Asn" residues. The &amp;, $*, d3 and $3 dihedral angles for each of the peptides are illustrated. </p><p>METHODS In this investigation, a total of ten peptides were examined with the general sequence of Leu-Ser-Arg- Leu-Phe-X-Y-AlaNH,, where X-Y is: ( 1) Asu"- Asn", (2) 3-amino-y-lactam"-Gly'z, (3) 4-amino-y- lactam"-Gly'2, (4) D-A1a1l-Prol2, ( 5 ) D-Pro'l-Asn'Z, (6) P-Ala"-Asn12, (7) L-Pro"-Gly'2, (8) ~-Aspl l - Asn',, (9) L-Ala"-Pro", (10) p-Asp"-Asnl2. Each of the peptides 1-6 is an active hypoglycaemic ana- logue of the hGH[6-13] peptide, while biological testing has shown that peptides 7-10 are inactive. </p><p>Molecular dynamics Energy calculations on the peptide series employed the CVFF potential force field. Using this force field, parameter assignments (bonds, valence angles, tor- sion angles, out-of-planes and 0-0 cross-terms) were made automatically (i.e. it was not necessary to make estimates of parameters: all were available as pre- determined). No Morse functions or cross-terms were used. An initial geometry for each peptide analogue was generated by building each of the sequences as an extended conformation using standard amino acid bond lengths, angles and sidechain dihedrals. Solvent was not included and all the amino acids were defined to be in their neutral states. All generated conformers were examined with the criterion employed that there was no high-energy cutoff, as higher-energy con- formations may be stabilised on binding to the recep- tor. Peptide bonds were forced to remain in the trans configuration by applying a torsional restraint of 10 kcal/mol rad2 during the calculations. </p><p>Energy minimisation of the starting structures was achieved using the steepest descents minimisation algorithm ( 16) followed by conjugate gradients minimisation (16) until a maximum derivative of </p></li><li><p>hGH[6-13] peptide analogues </p><p>approach followed in this investigation and to high- light the observed changes, these dihedral angles are plotted as a function of time (ps) in Fig. 3. Since conformers were collected at 1 ps intervals, the dihed- ral angles for each of the derived 100 conformers are given. The d2 dihedral angle shows two preferred angle ranges. The majority of conformers fall in the 42 dihedral angle range of - 70 to - 180", whilst a second smaller group appears approximately 30 ps into the trajectory and shows a 42 dihedral angle range of 40-70". The t+b2 dihedral angle shows a single angle range from - 100 to - 140, which is consistent with the restriction imposed on this bond by the a- aminosuccinimide ring structure. For the 43 dihedral angle two distinct ranges of -70 to -130" and 40-90" were observed. As there is only a single transition observed between the two conformer groups, this result indicates that there exists a consid- erable energy barrier to the interconversion. The $3 dihedral ranges (Fig. 3b) are 70-130" and -30 to - 80" and transitions between the conformers are </p><p>0.1 kcal/mol was reached. The MD run involved an initial equilibration at 1000 K for 1000 steps of 1 fs. Data were collected from a subsequent 1OOps MD run at 1000 K. This approach resulted in the genera- tion of 100000 conformers (dynamics steps of l fs) for each peptide analogue. Instantaneous dynamics structures were collected at 1 ps intervals giving a total of 100 conformers for each peptide analogue. For each of the conformers the temperature was then gradually reduced to 300 K over a period of 30 ps. Equilibration at 300 K was achieved using a 10 ps run of MD. For each of the peptide analogues 100 conformers were generated at 300 K, after which the conformer groups were minimised using the steepest descents method until the maximum derivative was less than 0.1 kcal/mol. This procedure was followed by quasi-Raphson-Newton minimisation until a max- imum derivative of lo-' kcal/mol was obtained. Minimisation had the effect of merely tightening the dihedral angle ranges, since it was found to be most efficient to minimise using the above protocol prior to sorting the conformers into groups. </p><p>Analysis of conformers Conformers were sorted based on the measured dihedral angles for those bonds in which some degree of freedom was possible. Along the backbone for peptides 1-5 and 7-9 a total of six flexible bonds are examined, while peptide 6 and 10 both incorporate the additional flexible bond, labelled p (Figs. 1 and 2). As our primary concern was the X-Y fragment, and conformations available to this moeity, the initial sorting of peptides was consequently based on the dihedral angles d2, t+b2, 43 and t+b3. Sorting of the dihedral angles resulted in the identification of a series of conformer families for each of the peptides. For a particular conformer group an average dihedral angle is reported along with the dihedral angle range. Only groups which include greater than 5% of the total conformer population were regarded as 'stable' conformations (17). On completion of the grouping procedure, other backbone and sidechain dihedral angles were examined, as was the likelihood of H bonding. A hydrogen bond (H bond) was considered highly probable if the prFton-acceptor distance did not exceed 0.25 nm (2.5 A) and the proton-acceptor angle was larger (1 8) than 135". </p><p>RESULTS AND DISCUSSION </p><p>Peptide 1 The structure of peptide 1 is shown in Fig. 1. High- temperature molecular dynamics of this peptide was followed by annealing, and finally energy minimis- ation, resulting in the collection of 100 conformers. The first point of interest evident from the analysis of these conformers relates the nature of the dihedral angles 42, t+b2, q53 and t,hg. To illustrate the general </p><p>0 20 40 60 80 </p><p>Trajectory time (psec) </p><p>b 1801 </p><p>- 120 2 F 60 </p><p>0 , o </p><p>-60 </p><p>m </p><p>0 m - 2 - m r O -120 </p><p>-180 1 0 20 40 60 80 </p><p>Trajectory time (psec) </p><p>FIGURE 3 The dihedral angles (a) d2 (+), GZ (0) and (b) d3 (a), c1/3 (+) as a function of the trajectory time (ps) for peptide 1. Each of the data points represents a single conformer which were collected at 1 ps intervals. </p><p>3 </p></li><li><p>K.A. Higgins et a1 </p><p>relatively frequent. Conformers were grouped based on dihedral angle values, and as a consequence nine separate conformer families were identified. </p><p>Table 1 provides a list of the 42, t,b2, 43 and t,b3 dihedral angle values for each of the conformer groups identified, along with the proportion of the total conformer population showing a particular con- formation. Seven of the nine conformers identified </p><p>TABLE 1 Conformer families arising from thr niolrciilor driiiimics mu!,, the </p><p>Asu-Asn resitliirs of prptiili, I </p><p>Conformer Dihedral angles ( ) ''4, conformer family </p><p>9 2 $ 2 4 3 $3 </p><p>6 </p><p>1 </p><p>1 </p><p>26 </p><p>a Each dihedral angle value represents the average. and the number in parentheses indicates the range 01' angles abailable for each of these dihedrals. </p><p>a Each dihedral angle value represents the average. and the number in parentheses indicates the range 01' angles available for each of these dihedrals. </p><p>for peptide 1 show an extended conformation. The other two (conformer groups 7 and 8) show a Type 11' 8-turn conformation based on the criteria of Lewis et (11. (19) and Capasso et al. (20). In addition each of the conformers making up the conformer group 7 fulfil the criteria (of distance and angle) for a H bond between the Phe CO and the Ala NH. In the Type 11' 8-turn conformation, a H bond distance consistent with a 1 +4 H bond is observed. </p><p>These findings with this peptide 1 are consistent with earlier X-ray crystallographic, circular dichroism and two-dimensional NMR studies (20-23) of CI- aminosuccinimide containing peptides which sug- gested 42 values of 55 and - 115", and t,b2 values limited to -120^. </p><p>Peptick 2 For peptide 2 conformers arising from the dynamics trajectory were treated in an identical manner to that discussed above for peptide 1. Initially the 42, t,b2, b3 and t,b3 dihedral angles were investigated. For the 42 dihedral angle two conformer groups were identified; one with a angle range of -80 to - 180", and the second with angles ranging from 30 to 70". The t,b2 angle was restricted due to the 3-y-lactam ring struc- ture with a single range of - 100 to - 140" observed. For the 43 dihedral angle, two conformer groups were identified with dihedral angles in the range 90-120" and -70 to - 110'. Transitions at the &amp; dihedral were more frequent with this peptide than observed for peptide 1. For the $3 dihedral angle two main conformer groups were identified with angle ranges of -90 to -40' and 50-90". </p><p>Sorting the conformers resulted in the identification of five separate conformer groups. Although the d 3 and t,b3 dihedra...</p></li></ul>