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