Membrane Protein Doppelgangers

Courtesy of Declan Doyle THE POTASSIUM TURNPIKE: KirBac1.1 in a membrane of a cell (green) ready to conduct potassium ions (yellow). Until recently, little progress has been made in determining high-resolution structural data for membrane proteins--the transporters, channels, and receptors that play critical roles in cell physiology. Now, scientists say they might be seeing the trickle that precedes the deluge. The current explosion of genomic information has led to the identification of prokaryotic homologs for many eukaryotic transporters and channels. These prokaryotic counterparts offer distinct advantages for crystallization, both in their diversity and ease of production. And while not every feature can be extrapolated to eukaryotes, the results could fill a few gaps in knowledge. "Membrane protein structure determination is taking off," writes Declan Doyle, University of Oxford, in an E-mail. Doyle and his group have solved the structure of the KirBac1.1 potassium channel from Burkholderia pseudomallei.1 This so-called inwardly rectifying channel protein allows unidirectional flow of potassium ions into cells, and it represents an important advance, says Doyle. "We have a more systematic approach that seems to be working." A SIMPLER SOLUTION Having a systematic approach could improve crystallization methods, which are still largely empirical. Researchers screen hundreds of experimental conditions, and membrane proteins require even more screening for the detergents that prevent these highly hydrophobic proteins from tangling. Such screening requires large quantities of proteins, which are most readily obtained through bacterial expression. Prokaryotic homologs also offer multiple structurally diverse targets, further enhancing chances of success. Researchers "pick the same general target, such as [ATP binding cassette, or ABC] transporters, [and] clone lots from different organisms," says Doyle. A pertinent example is the AcrB multidrug efflux pump, an ABC transporter from Escherichia coli.2 Human homologs of AcrB play a critical role in cancer chemotherapy and drug absorption. Using prokaryotic homologs allowed researchers to determine the first structure for a voltage-gated channel. Roderick MacKinnon, of Rockefeller University in New York, and his colleagues determined the structure of the KvAP potassium channel from Aeropyrum pernix, a thermophilic bacterium.3 This structure has provided important details and stirred significant controversy4 about the mechanism of voltage gating. This group also published the structure of a chloride channel from E. coli.5 Not all the recent advances in membrane protein structure owe their existence to genomic data; some represent long-term efforts. Ron Kaback, University of California, Los Angeles, and his colleagues recently published a structure for lactose permease from E. coli.6 This transporter has served for years as a model for the ion-coupled transporters that are so important in human physiology. Kaback attributes the success to using an inactive mutant with a "locked-in" conformation, thus overcoming the inherent flexibility of transporters, which works against crystallization. In other ongoing efforts, Columbia University's Jonathan Javitch has cloned about 20 bacterial homologs of neurotransmitter transporters, the targets of antidepressants, cocaine, and amphetamines in the brain. Others are attempting to express eukaryotic G-protein-coupled receptors, for which prokaryotic homologs have not been found, in E. coli.7 One potential difficulty in extrapolating results from prokaryotes to eukaryotes is the lack of posttranslational modification in bacteria. This deficit will impair the study of trafficking and regulation. But, Doyle says, the science won't suffer: "It will not place limitations on the interpretation." Javitch says that the lack of modifications such as glycosylation would actually "seem to be a good thing relative to the quite heterogeneous glycosylation one sees in eukaryotic systems." Despite these advances, the crystallization of any protein, membrane or soluble, still requires large amounts of protein and involves significant effort. Doyle and others say the methods are improving. Obtaining high-resolution structures represents the arduous but necessary next step towards understanding membrane proteins. According to Kaback, it's "the whole future." Megan M. Stephan (dr.meganstephan@att.net) is a freelance writer in Cheshire, Conn. References 1. A. Kuo et al., "Crystal structure of the potassium channel KirBac1.1 in the closed state," Science, 300:1922-6, June 20, 2003. 2. E.W. Yu et al., "Structural basis of multiple drug-binding capacity of the AcrB multidrug efflux pump," Science, 300:976-80, May 9, 2003. 3. Y. Jiang et al., "X-ray structure of a voltage-dependent K+ channel," Nature, 423:33-41, May 1, 2003. 4. G. Miller, "The puzzling portrait of a pore," Science, 300:2020-2, June 27, 2003. 5. R. Dutzler et al., "Gating the selectivity filter in ClC chloride channels," Science, 300:108-12, April 4, 2003. 6. J. Abramsonet al., "Structure and mechanism of the lactose permease of Escherichia coli," Science, 301:610-615, Aug 1, 2003 7. H.M. Weiss, R. Grisshammer, "Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli," Eur J Biochem, 269:82-92, 2002. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Membrane Protein Doppelgangers

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Courtesy of Declan Doyle
THE POTASSIUM TURNPIKE: KirBac1.1 in a membrane of a cell (green) ready to conduct potassium ions (yellow).