High Resolution Crystal Structures and Molecular Dynamics Studies Reveal Substrate Binding in the Porin Omp32

Abstract

The porin Omp32 is the major outer membrane protein of the bacterium Delftia acidovorans. The crystal structures of the strongly anion-selective porin alone and in complex with the substrate malate were solved at 1.5 and 1.45 Å resolution, respectively, and revealed a malate-binding motif adjacent to the channel constriction zone. Binding is mediated by interaction with a cluster of two arginine residues and two threonines. This binding site is specific for Omp32 and reflects the physiological adaptation of the organism to organic acids. Structural studies are combined with a 7-ns unbiased molecular dynamics simulation of the trimeric channel in a model membrane. Molecular dynamics trajectories show how malate ions are efficiently captured from the surrounding bulk solution by the electrostatic potential of the channel, translocated to the binding site region, and immobilized in the constriction zone. In accordance with these results, conductance measurements with Omp32 inserted in planar lipid membranes revealed binding of malate. The anion-selective channel Omp32 is the first reported example of a porin with a 16-stranded ␤-barrel and proven substrate specificity. This finding suggests a new view on the correlation of porin structure with substrate binding in specific channels.Porins are channel proteins that facilitate the exchange of ions and small hydrophilic substrate molecules across the outer membrane present in Gram-negative bacteria, mitochondria, and chloroplasts (1, 2). A number of bacterial porins have been studied structurally. They all possess ␤-barrels as the common structural principle (3). The trimeric bacterial porins are usually classified according to their number of barrelforming ␤-strands (3, 4); 18-stranded porins function as substratespecific channels and provide periplasmic binding proteins and uptake systems of the inner membrane with their specific substrates (5, 6), and 16-stranded porins are reported to merely act as unspecific ("general") diffusion pores, however, typically with a distinct ion selectivity (7-11).The current classification is mainly based on the crystal structures of 3 different 18-stranded and seven 16-stranded porins from Proteobacteria. Structural studies of substrate binding to trimeric porins were, thus, restricted to the sugar-specific maltoporins and to ScrY (5, 6). The nucleoside-specific pore protein Tsx and the fatty acid-binding transporter FadL are monomeric 12-and 14-stranded ␤-barrel proteins, respectively (17, 18). They are not regarded as porins proper; however, they also belong to the class of passive outer membrane channel proteins. Thus, in a more general view, substrate binding is not a special feature of 18-stranded outer membrane proteins. Several theories were developed to describe channel-substrate interactions which apply to diffusion pores in general (19). A decisive step of substrate translocation was modeled for maltoporin by conjugate peak refinement (20). Nonequilibrium molecular dynamics (MD) 2 and docking methods were applied to inves...

Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB

“…In PorB, three distinct translocation pathways appear to be housed within the same pore: one selective for sugars, one for cations, and one for anions. These results concur with the hypothesis that 16-stranded β-barrel OMPs have been mis-classified as nonspecific (36) and that porins may instead be multiply selective with sophisticated mechanisms for substrate selection.…”

“…1 B and C) identifies a putative pathway on the pore face nearest the crystallographic 3-fold axis that is lined with positively charged residues. A similar feature has been identified in the Escherichia coli OmpF (34,35), the Delfita acidovorans Omp32 (36,37), and the E. coli OmpC (38), which all share structural similarity to PorB in the transmembrane β-barrel region of the protein (SI Text and Fig. S3 A-C).…”

“…S3D). Site-directed mutagenesis (39)(40)(41), crystallographic analyses (36,42), and molecular dynamics simulations (35,36) suggest that the Lysine/ Arginine motif indeed acts as an anion translocation pathway. In PorB, the putative cation and anion translocation pathways do not overlap with the sugar-binding site or each other, suggesting that selective sugar transport and promiscuous cation and anion translocation through PorB are independent.…”

Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei

“…S1) (Zachariae et al, 2006). However, two additional b-strands were predicted in the endosymbiont protein, between residues 278-286 and 289-298, which have no corresponding motifs in Omp32, as shown in the comparative model ( Supplementary Fig.…”

“…A BLAST search against PDB revealed that the structure of the anion-selective trimeric porin Omp32 from Delftia acidovorans (PDB entry 2FGR) was the best template available (Zachariae et al, 2006), showing 47 % amino acid sequence similarity with the endosymbiont porin (see Supplementary Fig. S1).…”