Summary NHE9 (SLC9A9) is an endosomal cation/proton antiporter with orthologs in yeast and bacteria. Rare, missense substitutions in NHE9 are genetically linked with autism, but have not been functionally evaluated. Here we use evolutionary conservation analysis to build a model-structure of NHE9 based on the crystal structure of bacterial NhaA and use it to screen autism-associated variants in the human population first by phenotype complementation in yeast, followed by functional analysis in primary cortical astrocytes from mouse. NHE9-GFP localizes to recycling endosomes where it significantly alkalinizes luminal pH, elevates uptake of transferrin and the neurotransmitter glutamate, and stabilizes surface expression of transferrin receptor and GLAST transporter. In contrast, autism associated variants L236S, S438P and V176I lack function in astrocytes. Thus, we establish a neurobiological cell model of a candidate gene in autism. Loss of function mutations in NHE9 may contribute to autistic phenotype by modulating synaptic membrane protein expression and neurotransmitter clearance.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an unusual ABC transporter, functioning as a chloride channel critical for fluid homeostasis in multiple organs. Disruption of CFTR function is associated with cystic fibrosis making it an attractive therapeutic target. In addition, CFTR blockers are being developed as potential antidiarrheals. CFTR drug discovery is hampered by the lack of high resolution structural data, and considerable efforts have been invested in modeling the channel structure. Although previously published CFTR models that have been made publicly available mostly agree with experimental data relating to the overall structure, they present the channel in an outward-facing conformation that does not agree with expected properties of a "channel-like" structure. Here, we make available a model of CFTR in such a "channel-like" conformation, derived by a unique modeling approach combining restrained homology modeling and ROSETTA refinement. In contrast to others, the present model is in agreement with expected channel properties such as pore shape, dimensions, solvent accessibility, and experimentally derived distances. We have used the model to explore the interaction of open channel blockers within the pore, revealing a common binding mode and ionic interaction with K95, in agreement with experimental data. The binding-site was further validated using a virtual screening enrichment experiment, suggesting the model might be suitable for drug discovery. In addition, we subjected the model to a molecular dynamics simulation, revealing previously unaddressed salt-bridge interactions that may be important for structure stability and pore-lining residues that may take part in Cl(-) conductance.

Human NHA2 is a poorly characterized Na + /H + antiporter recently implicated in essential hypertension. We used a range of computational tools and evolutionary conservation analysis to build and validate a three-dimensional model of NHA2 based on the crystal structure of a distantly related bacterial transporter, NhaA. The model guided mutagenic evaluation of transport function, ion selectivity and pH dependence of NHA2 by phenotype screening in yeast. We describe a cluster of essential, highly conserved titratable residues located in an assembly region made of two discontinuous helices of inverted topology, each interrupted by extended chain. Whereas in NhaA, oppositely charged residues compensate for partial dipoles generated within this assembly, in NHA2, polar but uncharged residues suffice. Our findings led to a model for transport mechanism that was compared to the well known electroneutral NHE1 and electrogenic NhaA subtypes. This study establishes NHA2 as a prototype for the poorly understood, yet ubiquitous, CPA2 antiporter family recently recognized in plants and metazoans and illustrates a structure-driven approach to derive functional information on a newly discovered transporter.

Structural genomics initiatives provide ample structures of "hypothetical proteins" (i.e., proteins of unknown function) at an ever increasing rate. However, without function annotation, this structural goldmine is of little use to biologists who are interested in particular molecular systems. To this end, we used (an improved version of) the PatchFinder algorithm for the detection of functional regions on the protein surface, which could mediate its interactions with, e.g., substrates, ligands, and other proteins. Examination, using a data set of annotated proteins, showed that PatchFinder outperforms similar methods. We collected 757 structures of hypothetical proteins and their predicted functional regions in the N-Func database. Inspection of several of these regions demonstrated that they are useful for function prediction. For example, we suggested an interprotein interface and a putative nucleotide-binding site. A web-server implementation of PatchFinder and the N-Func database are available at http://patchfinder.tau.ac.il/.

The copper-transporting ATPase ATP7B has an essential role in human physiology, particularly for the liver and brain function. Inactivation of ATP7B is associated with a severe hepato-neurologic disorder, Wilson disease (WD). Hundreds of WD related mutations have been identified in ATP7B to date. The low frequency and the compound-heterozygous nature of causative mutations complicate the analysis of individual mutants and the establishment of genotype-phenotype correlations. To facilitate studies of disease-causing mutations and mechanistic understanding of WD, we have homology-modelled the ATP7B core (residues 643-1377) using the recent structure of the bacterial copper-ATPase LCopA as a template. The model, supported by evolutionary conservation and hydrophobicity analysis, as well as existing and new mutagenesis data, allows molecular interpretations of experimentally characterized clinical mutations. We also illustrate that structure and conservation can be used to grade potential deleterious effects for many WD mutations, which were clinically detected but have not yet been experimentally characterized. Finally, we compare the structural features of ATP7B and LCopA and discuss specific features of the eukaryotic copper pump.