August Krogh Institute, University of Copenhagen Staff information

Peter Leth Jørgensen

Current research projects

Biomembrane Research Center, August Krogh Institute, University of Copenhagen

In our part of the Biomembrane Center we apply DNA technology, molecular biological and cell physiological methods to study the structure and molecular mechanism of the Na,K-pump and other Na,K-transport proteins. We also attempt to integrate this knowledge to understand the regulation and physiological function of active Na,K-transport in kidney and other organs.

Staff of the Biomembrane Center at August Krogh Institute

Professor dr.med.sci. Peter L. Jørgensen
Associate professor lic. techn. Per Amstrup Pedersen
Assistant professor: N.N.
Ph.D. student, cand.scient. Jesper M. Nielsen
Ph.D. student, cand.scient. Jakob H. Rasmussen
Ph.D. student, cand.scient. Karl Lücking
Ph.D. student, cand.scient. Bo Kristensen
Undergraduate students: Jesper Jørgensen, Mette A. Jacobsen, Jens H.Larsen, Allan Røske Nielsen.
Laboratory technicians: David Sørensen, Lilian B. Holgersen, Dorthe Meinertz.
Secretary: Kirsten Menne

Structure and molecular mechanism of Na,K-ATPase

In our working hypothesis for the transport mechanism of the Na,K-pump, ATP drives shuttling between the occluded E2[2K] and E1P[3Na] conformations with binding and release of Na+ and K+ in a ping pong sequence. It is therefore important to identify amino acid side chains engaged in ATP binding, cation occlusion and E1-E2 transitions. To understand the cation exchange mechanism it is of particular interest to identify amino acid residues that are alternately engaged in coordination of Na+ and K+ in the two occlusion cavities.

Our project is divided in two main parts:

A. Three-dimensional structure of the Na,K-pump

Crystallization of the Na,K-pump proteins to solve the 3-dimensional structure at high resolution. Attempts to crystallize the purified renal Na,K-ATPase have so far not been successful. As an alternative we also attempt to crystallize the large ATP binding central domain (residues 340-776) of the a-subunit after expression i E.coli.

B. Heterologous expression and site directed mutagenesis.

To establish structure function relationships by site-directed mutagenesis we found it important to develop a system for heterologous expression in yeast (P.A. Pedersen et al. J.Biol.Chem. 271: 2514, 1966) that allows assay of ligand binding to the recombinant protein at equilibrium, in the absence of contaminant endogenous Na,K-ATPase.

ATP binding at equilibrium

ATP binding to wild type yeast Na,K-ATPase and the mutant Asp369Ala allowed estimation of the expense in free energy required to position the g-phosphate in proximity of the carboxylate group of Asp369 and the role of this residue in governing long range E1-E2 transitions. An arginine residue (Arg546) is bordering a hydrophobic pocket for nucleotide binding in a-subunit of Na,K-ATPase.

Potassium ion occlusion at equilibrium

Assay of Rb+ and Tl+ occlusion to recombinant Na,K-ATPase from yeast were developed to establish structure function relationships of the amino acids that may form the cavity for occlusion of K+ in the E2[2K]-form. Wild type yeast enzyme was capable of occluding two Rb+ or Tl+-ions per ouabain binding site or a 1b 1-unit with high apparent affinity (Kd(Tl+) = 7 ± 2 mM), like the purified Na,K-ATPase from pig kidney. High affinity occlusion of Rb+ or Tl+ ions was abolished by mutations to Glu327(Gln,Asp), Asp804(Asn,Glu), Asp808(Asn,Glu) and Glu779(Asp). The substitution of Glu779 for Gln reduced the occlusion capacity to one Tl+ ion per a 1b 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 ± 8 mM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+-displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 are therefore essential for high affinity occlusion of K+ in the E2 [ 2K]-form.

Na-dependent phosphorylation

Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Glu327, Glu779, Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[ 3Na] form. Demonstration of alternate interactions of Na+ or K+ with these carboxylate residues support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.

Teaching

Undergraduate students in biology or biochemistry: The semester course in molecular biology with experimental course in gene technology and the ph.d. course in heterologous expression at AKI are based on the methods we have developed in the research center. The staff at the centre also teaches at courses in protein structure and function, general physiology and human physiology.

In addition the staff supervises a number of Ph.D. students.

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August Krogh Institute | Last update on October 30, 1997 | Comments to pljorgense@aki.ku.dk