Publications

2002

Yi, S., Bernat, B., Pál, G., Kossiakoff, A. & Li, W. H.

Functional promiscuity of squirrel monkey growth hormone receptor toward both primate and nonprimate growth hormones.

Mol Biol Evol 19, 1083-92.

Primate growth hormone (GH) has evolved rapidly, having undergone approximately 30% amino acid substitutions from the inferred ancestral eutherian sequence. Nevertheless, human growth hormone (hGH) is physiologically effective when administered to nonprimate mammals. In contrast, its functional counterpart, the human growth hormone receptor (hGHR), has evolved species specificity so that it responds only to Old World primate GHs. It has been proposed that this species specificity of the hGHR is largely caused by the Leu --> Arg change at position 43 after a prior His --> Asp change at position 171 of the GH. Sequence analyses supported this hypothesis and revealed that the transitional phase in the GH:GHR coevolution still persists in New World monkeys. For example, although the GH of the squirrel monkey has the His --> Asp substitution at position 171, residue 43 of its GHR is a Leu, the nonprimate residue. If the squirrel monkey truly represents an intermediate stage of GH:GHR coevolution, its GHR should respond to both hGH and nonprimate GH. Also, if the emergence of species specificity was a result of the selection for a more efficient GH:GHR interaction, then changing residue 43 of the squirrel monkey growth hormone receptor (smGHR) to Arg should increase its binding affinity toward higher primate GH. To test these hypotheses, we performed protein-binding assays between the smGHR and both human and rat GHs, using the surface plasmon resonance methodology. Furthermore, the effects of reciprocal mutations at position 43 of human and squirrel monkey GHRs are measured for their binding affinities toward human and squirrel monkey GHs. The results from the binding kinetic assays clearly demonstrate that the smGHR is in the intermediate state of the evolution of species specificity. Interestingly, the altered residue Arg at position 43 of the smGHR does not lead to an increased binding affinity. The implications of these results on the evolution of the GH:GHR interaction and on functional evolution are discussed.

Wakelin, S., Conibear, P. B., Woolley, R. J., Floyd, D. N., Bagshaw, C. R., Kovács, M. & Málnási-Csizmadia, A.

Engineering Dictyostelium discoideum myosin II for the introduction of site-specific fluorescence probes.

J Muscle Res Cell Motil 23, 673-83.

Dictyostelium discoideum is a useful host for the production of constructs for the analysis of structure-function relationships of myosin. Here we describe the use of myosin II constructs containing a single tryptophan residue, at different locations, for probing events at the nucleotide binding site, the relay loop and the communication path between them. GFP fusions have also been expressed at the N- and C-termini of the myosin motor to provide sensitive probes of the actomyosin dissociation reaction in microscope-based kinetic assays. We report on the fluorescence anisotropy of these constructs in the context of their use as resonance energy transfer probes.

Reményi, A., Tomilin, A., Scholer, H. R. & Wilmanns, M.

Differential activity by DNA-induced quarternary structures of POU transcription factors.

Biochem Pharmacol 64, 979-84.

Recent structural studies on transcription factors from the POU family in complex with multiple cognate DNA enhancer elements have established a novel concept in DNA-mediated formation of distinct conformations of transcription regulator assemblies. Two crystal structures of the Oct-1 transcription factor in the presence of two different DNA sites have demonstrated how its POU DNA-binding segment is capable in forming two unrelated dimer arrangements, which is DNA motif dependent. While one arrangement allows binding of the Oct-1 specific coactivator OBF-1, binding of this coactivator is blocked in the second arrangement because the binding site is involved in its own dimer assembly. Conversely, two crystal structures of another POU transcription factor, Pit-1, have demonstrated how the same overall assembly is maintained in the presence of two different DNA response elements. However, since the distance of the two Pit-1 half-binding sites on these elements differ by two base pairs, the overall dimensions of the two complexes vary, allowing binding of a specific represssor (N-CoR) in one conformation but not in the other. Thus, despite the occurrence of different DNA-mediated molecular mechanisms, the net result, conformation-dependent binding of further regulators, is equivalent. These data introduce a concept where the DNA motif not only serves as binding site for specific transcription factors but also regulates their function by mediating specific transcription factor assemblies, which determine binding to conformation-dependent coregulators.

Patthy, A., Amir, S., Malik, Z., Bódi, A., Kardos, J., Asbóth, B. & Gráf, L.

Remarkable phylum selectivity of a Schistocerca gregaria trypsin inhibitor: the possible role of enzyme-inhibitor flexibility.

Arch Biochem Biophys 398, 179-87.

Kukor, Z., Tóth, M., Pál, G. & Sahin-Tóth, M.

Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation.

J Biol Chem 277, 6111-7.

Mutation of Arg(117), an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg(117) was investigated in wild-type cationic Tg and in the activation-resistant Lys(15) --> Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg(117) can be examined selectively. We found that trypsinolytic cleavage of the Arg(117)-Val(118) bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca(2+), at pH 8.0, the hydrolysis equilibrium (K(hyd) = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca(2+) reduced the rate of cleavage at Arg(117) at least 20-fold, and shifted K(hyd) to 0.7. These observations indicate that the Arg(117)-Val(118) bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K(i) of 80 microm, while no inhibition was observed with K15Q-Tg carrying the Arg(117) --> His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg(117). Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg(117) results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg(117)-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg(117) --> His mutation.

Kovács, M., Málnási-Csizmadia, A., Woolley, R. J. & Bagshaw, C. R.

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site.

J Biol Chem 277, 28459-67.

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.

Kim, E., Bobkova, E., Hegyi, G., Mühlrad, A. & Reisler, E.

Actin cross-linking and inhibition of the actomyosin motor.

Biochemistry 41, 86-93.

Katona, G., Berglund, G. I., Hajdu, J., Gráf, L. & Szilágyi, L.

Crystal structure reveals basis for the inhibitor resistance of human brain trypsin.

J Mol Biol 315, 1209-18.

Gáspári, Z., Patthy, A., Gráf, L. & Perczel, A.

Comparative structure analysis of proteinase inhibitors from the desert locust, Schistocerca gregaria.

Eur J Biochem 269, 527-37.

The solution structure of three small serine proteinase inhibitors, two natural and one engineered protein, SGCI (Schistocerca gregaria chymotrypsin inhibitor), SGCI[L30R, K31M] and SGTI (Schistocerca gregaria trypsin inhibitor), were determined by homonuclear NMR-spectroscopy. The molecules exhibit different specificities towards target proteinases, where SGCI is a good chymotrypsin inhibitor, its mutant is a potent trypsin inhibitor, and SGTI inhibits both proteinases weakly. Interestingly, SGTI is a much better inhibitor of insect proteinases than of the mammalian ones used in common assays. All three molecules have a similar fold composed from three antiparallel beta-pleated sheets with three disulfide bridges. The proteinase binding loop has a somewhat distinct geometry in all three peptides. Moreover, the stabilization of the structure is different in SGCI and SGTI. Proton-deuterium exchange experiments are indicative of a highly rigid core in SGTI but not in SGCI. We suggest that the observed structural properties play a significant role in the specificity of these inhibitors.