HAMP domains connect extracellular sensory with intracellular signaling domains in over 7500 proteins, including histidine kinases, adenylyl cyclases, chemotaxis receptors, and phosphatases. The solution structure of an archaeal HAMP domain shows a homodimeric, four-helical, parallel coiled coil with unusual interhelical packing, related to the canonical packing by rotation of the helices. This suggests a model for the mechanism of signal transduction, in which HAMP alternates between the observed conformation and a canonical coiled coil. We explored this mechanism in vitro and in vivo using HAMP domain fusions with a mycobacterial adenylyl cyclase and an E. coli chemotaxis receptor. Structural and functional studies show that the equilibrium between the two forms is dependent on the side-chain size of residue 291, which is alanine in the wild-type protein.

Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-prolinealanine (NPA) motifs, the central constriction, and an aromatic͞ arginine (ar͞R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar͞R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A͞ R195V), did not affect water permeability. The double mutant AQP1-H180A͞R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A͞H180A) enlarged the maximal diameter of the ar͞R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar͞R constriction in AQP1-R195V and AQP1-H180A͞R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar͞R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar͞R constriction. mutational analysis ͉ proton filter ͉ solute selectivity O rthodox aquaporins (AQPs) constitute one branch of water-conducting channels within the superfamily of major intrinsic proteins (1). In recent years, the protein structure of the prototypical mammalian AQP1 has been refined to 2.2-Å resolution (2-5). Two highly conserved structural features within the channel were proposed as filters that exclude the passage of solutes larger than water and of charged molecules, including protons. A central constriction is formed by the capping amino acids Asn-Pro-Ala (NPA constriction) at each positive end of two short ␣-helices, such that the two NPA motifs are pinched in the center of the pore. Proline and alanine are exchangeable to some extent, whereas asparagine is invariable (1). Extensive molecular dynamics͞quantum mechanical simulations suggest that the free energy barrier located at the NPA constriction predominates in the exclusion of protons (reviewed in ref. 6). Depending on the computational approach, a second significant energy barrier, termed aromatic͞arginine (ar͞R) constriction, exists. It is located below the channel mouth and is even narrower than the central NPA constriction (6, 7). It is formed by four amino acids (Phe-56, His-180, Cys-189, and Arg-195 in rat AQP1). The ensemble of Phe, His, and Arg is highly conserved in orthodox AQPs. Their side chains directly flank the pore, whereas the less-conserved C...

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The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis.Malaria research profits tremendously from the efforts in genomic data acquisition from the Plasmodium sp. parasite. More than 27 megabases of genomic nucleotide information were available by October 2000 covering the 14 chromosomes to at least 90% (1). Now, chromosomes 2, 3, and 5 are complete; the others are in the final, gap closing stage. Using genome data, Woodrow et al. (2) identified a high affinity hexose transporter that allows the parasite to take up its main energy source, glucose, from the host serum. Surolia and Surolia (3) found a key enzyme of a de novo glycerolipid synthesis pathway in the P. falciparum genome and showed its inhibition by triclosan. Until then, the parasite was thought to be fully dependent on the uptake of lipid precursors from the serum (4).The massive synthesis of phospholipids is a hallmark in the metabolism of blood-stage parasites be it from precursors or de novo. As soon as the free parasite in its merozoite form has invaded an erythrocyte it begins a series of major transformations accompanied by rapid growth (5). In the ring stage, it starts to modulate the ionic composition of the red cell cytoplasm and a cytoadhesive protein (PfEMP1) with high affinity to blood vessel endothelia is inserted into the red cell membrane leading to sequestration (6). The next, trophozoite stage is characterized by the highest rates of metabolism and growth (7). Later, as schizonts, the parasite prepares its asexual multiplication by DNA replication. Finally, the red cell bursts and releases new schizont-derived merozoites to start another cycle. This 48-h process produces up to 32 merozoites, which means that the parasite has to multiply the amount of membrane lipids enormously (8). The parasite is devoid of a pathway for cholesterol biosynthesis. All newly synthesized membrane lipids are of the glycerolipid variety, ...