Structure and Function of Peptide Hormones and Peptide Hormone/Receptor Interactions

Peptide hormones play an essential role in most physiological regulation mechanisms. The interaction with their membrane bound receptors has to be highly specific to result in signal transduction into the cell. Little is known about the structure of peptide hormone/ receptor complexes, and structure determination of such complexes is still a challenge in structural biology. From structure determination of isolated peptide hormones together with biochemical and biophysical data, however, indirect information about the mode of receptor interaction and the receptor bound structural state of the hormone can be deduced. Therefore, functional and structural studies of peptide hormones are expected to shed further light on the pathology and possible therapy of several endocrine diseases. We also focus on direct studies of peptide hormone/receptor interactions using soluble fragments of the respective receptor.


Parathyroid hormone (PTH) and PTH-Receptor

PTH is involved in blood calcium homeostasis and bone metabolism, and its medical importance is based on its role in the therapy of osteoporosis. Two helices have been identified for PTH fragments with intact NH2 terminus (Marx et al., 1995, 2000, 2001). Truncation of the two NH2-terminal amino acid residues, however, leads to a complete loss of in vivo normocalcemic activity. In order to elucidate a structure-activity relation we have determined the three-dimensional structures of the NH2-terminally truncated PTH-fragments hPTH(2-37), hPTH(3-37), hPTH(4-37) using NMR spectroscopy. The two inactive fragments hPTH(3-37) and hPTH(4-37) do not feature the NH2-terminal helix anymore, in contrast to the biological active fragments hPTH(1-37) and hPTH(2-37). From these results we conclude that the presence of the NH2-terminal helix is correlated with the in vivo normocalcemic function of PTH (Marx et al., 1998a, 2001). As a first step in the design of a synthetic drug we have shown that the NH2-terminal helix can be restored by acetylation or succinylation on the NH2 terminus of the truncated peptides (Marx et al., 2001).
The peptide hormones hPTH(1-34) and hPTHrP(1-34) (hPTHrP = human PTH-related protein) show similar secondary structures under near physiological solution conditions but differ in the middle part of the peptides: hPTH(1-34) shows hydrophobic interactions resulting in a definded loop region, whereas PTHrP(1-34) does not exhibit such tertiary interactions in near physiological solution. As the two peptides bind to and activate the same receptor, one of them is expected to undergo a structural rearrangement upon binding. Such a rearrangement can be induced for hPTH(1-34) on addition of 20% TFE, resulting in a stabilization of the two helices and in the lost of the hydrophobic interactions. Under the assumption that TFE mimicks a membrane like environment, a more open conformation, like that of hPTHrP(1-34) already under near physiological conditions, can be suggested for the receptor bound state (Weidler et al., 1999; Marx et al., 2000).

... Preliminary studies on the structure of fragments of the PTH-Receptor were already done, too. ...

To be continued...

For further information on the current research in this field do not hesitate to contact  either  PD Dr. Birgitta Wöhrl or Prof. Dr. Paul Rösch.



The Guanylin and Uroguanylin / Guanylatcyclase-System

Infection with enterotoxigenic strains of E. coli are a main cause of infant mortality in many developing countries and also of traveler's diarrhea. These bacteria produce peptidogenic enterotoxins that bind to the receptor guanylyl cylase-C (GC-C) found in the microvillus membranes of the intestine and stimulate uncontrolled fluid secretion by hyperactivation of this receptor. The heat-stable enterotoxin STa from E. coli is known for such an effect. The search for potential endogenous ligands of GC-C led to the discovery of the peptide hormones guanylin and uroguanylin, which possess a high degree of sequence similarity with heat-stable enterotoxins. Guanylin and uroguanylin are short peptides containing four cysteines, which are disulfide linked in a 1-3/2-4 pattern. The correct disulfide connectivity is essential for the biological activity of both peptides (Klodt et al., 1997) and gives rise to the existence of two topological isomers, each, in an approximately 1:1 ratio as demonstrated by HPLC chromatography and NMR spectroscopy (Adermann et al., 1996; Klodt et al., 1997; Marx et al., 1998b, 2001; Schulz et al., 1998). We have shown, that only one of these interconvertible isoforms meets the structural requirements to bind to and activate GC-C (Marx et al., 1998b, 2001; Schulz et al., 1998). The three-dimensional structure of this bioactive isomer (isomer A) closely resembles the crystal structure of STa by Ozaki et al. (Marx et al., 1998b).
In order to investigate the influence of certain amino acid side chains on the isomerism and on the biological activity of guanylin and uroguanylin peptides, synthetic derivatives of uroguanylin and guanylin (L-alanine scan as well as chimeric peptides) were compared by HPLC, 2D 1H NMR spectroscopy and by their GC-C activating potency (Schulz et al., in press). These studies approve our interconversion model (Marx et al., 2001) and confirm that the conformational conversion is predominantly under the control of a COOH-terminal sterical hindrance. Furthermore, the mutant peptides show drastical variations in their biological activity. NMR chemical shift analysis revealed that the isomers of the derivatives exhibit structures similar to that of the respective wild-type peptide. These results point to selective amino acid side chains that are involved in receptor binding and activation.

For further information on the current research in this field do not hesitate to contact  either  PD Dr. Ute C. Marx or Dr. Klaus Vitzithum.





Guanylin is mainly secreted as the corresponding prohormone containing 94 amino acid residues, with the mature hormone located at its very COOH terminus. In this molecular prohormone form, human proguanylin exhibits only negligible GC-C activating potency (Schulz et al., 1999). The role of the prosequence for the proper folding of guanylin and for the missing bioactivity of the prohormone compared to the mature hormone can be explained on a structural level, as the termini of the prohormone interact with each other forming a triple stranded b-sheet, and therefore, shieldin g the hormone region from receptor interactions. The three-dimensional structure of the guanylin-prohormone also provides implications for the peptide hormone/ receptor interaction. In its prohormone form the sequence corresponding to guanylin is fixed in its A isomer topology (Lauber et al., 2003a). From our model structure of the extracellular domain of GC-C the geometry of the ligand binding sequence is predicted to be located close to an exposed and accessible b-strand. Since in its prohormone form guanylin binds to the NH2-terminal residues by forming an intramolecular triple-stranded b-sheet (Lauber et al., 2003) it is possible that a similar kind of interaction is involved in the binding of guanylin to its receptor in addition to specific side chain interactions.
For the structure determination of proguanylin it was necessary to confirm its monomeric state and to develop a bacterial expression system for uniform 15N and 13C isotopic-labelling. When producing recombinant proteins correct folding for accurate disulfide formation is a matter of particular interest. Thus, for the expression and isotopic-labelling of proguanylin and other disulfide bridged proteins (e.g.: domain 1 and 15 of LEKTI, miniGC-C) we have developed an expression strategy based on E. coli strains with oxidative cytoplasm (Lauber et al., 2001, 2002). The oligomerisation state of proguanylin was determined using analytical ultracentrifugation (Lauber et al., 2002).

For further information on the current research in this field do not hesitate to contact  either  PD Dr. Ute C. Marx or Dr. Klaus Vitzithum.




Peptide Hormone Receptor Interactions:
The Guanylin(Uroguanylin/STa)/ Guanylyl Cyclase-C System

With the investigation of the guanylin(uroguanylin/STa)/ guanylyl cyclase-C system we will contribute to the growing research field of membrane bound receptors and to the understanding of the principles of peptide hormone/ receptor recognition. In addition, this peptide hormone/ receptor system bears a high pharmacological potential as infection with enterotoxic E. coli strains is a main cause of infant mortality in developing countries. Spectroscopical studies shall reveal molecular causes responsible for ligand binding affinity and specificity as well as for receptor activation on a structural level. For this purpose soluble and properly folded fragments of the extracellular domain of guanylyl cyclase C (GC-C) will be investigated. The interactions of two fragments (197 and 407 residues) of GC-C with the endogenous ligands guanylin and uroguanylin as well as the heat-stable enterotixin STa from E. coli will be studied by various biophysical methods including NMR spectroscopy.

For further information on the current research in this field do not hesitate to contact  either  PD Dr. Ute C. Marx or Dr. Klaus Vitzithum.





To be continued...