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Etana Padan
Etana Padan
Synopsis of Research:

(The references are cited according to the List of Publications)
                                                                                                                                  
The research subjects of Etana Padan are in molecular and structural biology of membrane proteins. These topics are in the front of modern biochemistry. Membrane proteins represent about 30 % of the proteome and have indispensable functions in all living cells; they are involved in energy storage and transduction, transport of solutes into the cells and export of toxic material from the cells and signal transduction across membranes, an essential process in cell communication and normal proliferation. It is not surprising, that about 60% of the drug targets are membrane proteins.
The group of Etana Padan discovered anoxygenic photosynthesis in cyanobacteria (16,17,21-23,25,27,28,29,31,32,41,48,49,53,54,61,64,66,67) and the responsible enzyme, sulfide quinone oxido-reductase (SQR) (70,76,96,126), an integral membrane protein (in Oscillatori limnetica) . Her group cloned the gene, expressed and purified the protein and characterized its properties. These achievements have been a landmark in the understanding of the physiology and ecology of prokaryotic phototrophs; SQR-based photosynthesis has since been shown to be a general property of many prokaryotic phototrophs and to have a paramount ecological importance in niches in which anoxic conditions prevail (41,83,86,88,101,104,110,116). Interestingly, recently SQR has been found in other bacteria and even in yeast mitochondria, suggesting a role in detoxification of sulfide in anaerobic eukaryotes.
The present main research of Etana Padan is the NhaA Na+/H+ antiporter of Escherichia coli, an integral membrane protein that exchanges H+ and Na+ across the membrane and is involved in homeostasis of pH and Na+, a requisite of all living cells. Homeostasis of pH, its physiological importance and the primary role of Na+/H+ antiporters in this basic cellular process were first shown in Escherichia coli by the group of Padan (20,30,38,43,44,46,51,52,58,59,60,62,115,139,142,145). Similar antiporters are ubiquitous throughout the biological world. In humans, for example, their controlled activity is critical to avoid heart failure conditions. In plants they form the basis of engineering salt resistant plants. Recently, the genome project showed that orthologs of NhaA antiporter exist from bacteria to humans. The current focus of the project of E. Padan is to understand the relationship between the structure of this integral membrane protein and its function in pH and Na+ homeostasis. For this purpose, the group of E. Padan cloned the nhaA gene (63,68,75) discovered its unique Na+ dependent regulation (78,81,97,98,100,102,107,108,113,115,119,124,127), over-expressed the protein in a functional form and revealed the properties that underpin its role in pH and Na+ homeostasis (77,82,84,85,89,145). It is an electrogenic antiporter that exchanges two protons per one Na+ (90,146). Similar to many other antiporters it is tightly regulated by pH, a property essential for pH homeostasis (91,94,95,98,105-107,109,111,112,120,122,123,130,133,134,135,136-138). This achievement led to a very tight collaboration between the group of E. Padan and the group of H. Michel (Max Planck for Biophysics, Frankfurt, Germany). In this combined effort (121,125,128,129,131,132) the crystal structure of NhaA has recently been solved. A new fold of a membrane protein has been unraveled. This most significant work has recently been published in a Nature article (141) followed by in-silico electrostatic analysis of NhaA, a novel approach in secondary transporters (147).
It should be emphasized that while the crystal structure of 30000 soluble proteins are known, only about 90 structures of membrane proteins have been determined and these include only seven transport proteins. NhaA is the first structure of a Na+/H+ antiporter. Therefore, these achievements of E. Padan provide two major insights: the first is in the understanding of the architecture, mechanism of ion transport and regulation of Na+/H+ antiporters, most essential transporters in all living cells including humans and higher plants; the second is in the general understanding of the architecture and function of integral membrane proteins.
                                                                                                                                
Remarkably, recently, the crystal structure of several other ion coupled transporters have been determined, revealing a novel fold of inverted topology repeates similar to that of NhaA. Each repeat contains a trans membrane segment interrupted by  mid-membrane extended-chain. Relative to α-helices, interrupted helices by extended chains may confer flexibility at lower energy cost and allow the conformational changes needed for the alternate accessibility mechanism of ion transport.
 Based on the crystal structure of NhaA, novel research approaches have been opened to study the antiporter.
a. In-silico electrostatic analysis and Molecular Dynamics (MD) simulations of NhaA, novel approaches in secondary transporters were applied (146, 149,157). It revealed the unique electrostatic interactions within the protein that are crucial for its activity and the influence of protonation states on the dynamics of the protein.  b. Structural-based evolutionary computational method combined with biochemistry identified and localized pH induced conformational changes involved in the pH regulation of NhaA (148,121,125,154,155,160). c. Many transporters and channels including NhaA (123,143,150,153) exist in the native membrane as oligomers. In most cases, the functional/structural role of the oligomeric state is still elusive. The crystal structure of NhaA (141, 147) allowed to design and construct a new mutant of NhaA that encodes a monomeric NhaA in the native membrane (151). Comparison of the monomer properties to those of the dimer has revealed that the monomer is the functional unit of NhaA (151, 155). However, the dimer is essential for adaptation to extreme stress conditions with respect to pH and Na+ (156, 161). d. Direct clinical importance is apparent since the Na+ cycle participates in the virulence of several pathogenic enterobacteria. Padan and colleugues demonstrated the importance of NhaA in the life cycle of Vibrio cholerae (134). Based on the crystal structure and using bioinformatics Padan and colleugues have recently modeled NHE1 (152), the house keeping  human Na+/H+ antiporter that plays primary role in heart conditions in humans and exhibits a pH control phenomenon similar to NhaA. The model supported by extensive mutagenesis work is the first step toward rational drug design. Modeling of the NHA2, a human NhaA orthologue suggested to be an essential hypertension factor is also currently in progress.  
Summary
It should be emphasized that while the crystal structure of 30000 soluble proteins are known, only about 90 structures of membrane proteins have been determined and these include only ten structures of transport proteins. Thus, lack of crystal structures is a major bottle neck in drug design to transporters targets. The crystal structure of NhaA is the first and only structure of a Na+/H+ antiporter. High lighted by the crystal structure the other experimental achievements of E. Padan together with the crystal structure have become a paradigm of antiporter study and also bear upon the general architecture of ion coupled transporters as summarized below: 1. The crystal structure of NhaA has revealed the structure/function/regulation relationship of an antiporter. 2. The crystal structure opened new avenues of research combining computation and experimental approaches crucial for the understanding of the mechanism of Na+/H+ exchange. 3. The inverted topology repeat with mid-membrane interrupted helices found in NhaA and shown to be common in other ion coupled transporters has been suggested the structural basis of the alternate access–mechanism of ion transport.  4. New perspective on the classification of membrane proteins is emerging: it is difficult if not impossible, to predict internal structural repeats from the gene sequence. Members of different gene families with similar functions may have homologous 3D structures and therefore a common architecture implies a common transport mechanism  5. Understanding the mechanism of NhaA and its regulation provides a clue to the role of NhaA-type antiporters in the adaptive response of pathogenic enterobacteria to H+ and Na+ stresses which occure during their life cycle and bear upon their pathogenicity. 6. Importance in drug design has become apparent; using NhaA structure as a template models of human NHE1 and NHA2  Na+/H+ antiporters have been achieved. NHE1 plays a primary role in heart conditions while NHA2 has been suggested a candidate transporter in essential hypertension in humans. Remarkably, increasing number of bacterial transporters have been shown to have human orthologues and because the bacterial transporters are more amenable to crystallization they serve as model systems to the study of most important human transporters. These include Na+/H+ antiporters, neurotransmitter transporters and others. 7. Understanding the mechanism of a pH-controlled transporter has an obvious biotechnological merit.