Structure and Function of Pore-Forming b-Barrels from Bacteria

Crystallographic studies of the past ten years have revealed that many outer membrane proteins and bacterial toxins are constructed on the b-barrel motif. Two structural classes can be identified. The first class, represented by the porins, includes monomeric or multimeric proteins where each b-barrel is formed from a single polypeptide. The second class features proteins where the b-barrel is itself a multimeric assembly, to which each subunit contributes a few b-strands. In addition to structural investigations, much work has also been devoted to the functional aspects of these proteins, and to the relationships between structure and function. Here we present a review of the structural and the functional properties of some of the beststudied examples of these various classes of proteins, namely the general-diffusion, specific and ligand-gated porins, multidrug efflux proteins and the staphylococcal toxin a-hemolysin. The b-barrel is a common feature for integral membrane proteins of bacterial origin. In particular, proteins involved in transport across bacterial outer membranes share this structural motif. Much attention has recently been devoted to these proteins, as atomic resolution structures have flourished in the past few years (Table). The reader is directed to recent reviews dealing with the more structural aspects of the research carried out on these proteins (Schirmer, 1998; Buchanan, 1999; Koebnik et al., 2000; Schultz, 2000). Although, the discussion of some structural features is unavoidable here, this review will be centered on the more functional or dynamic aspects of these proteins. The last couple of years have seen an explosion of articles on porins from a great variety of microorganisms. For sake of conciseness, this review is essentially focused on Escherichia coli, with mention of a handful of other organisms. This somewhat slanted view is not meant to mitigate the importance of other studies. General Diffusion Porins Members of the general diffusion porin family, such as the Escherichia coli OmpF, OmpC and PhoE proteins and the Klebisiella pneumoniae OmpK36 (Figure 1), are examples of the best characterized b-barrel channels at the functional level. Still, the molecular basis and physiological relevance of many of their properties, such as voltage-dependence, cooperativity and modulation, have remained elusive. These proteins form trimers in the outer membrane of Gram-negative bacteria, where they play the major role of molecular sieves. They allow for the fast flow of hydrophilic solutes of molecular weight less than 600 through the highly hydrophobic protective barrier formed by the outer membrane and its lipopolysaccharide layer (Nikaido, 1996). A major breakthrough in porin research occurred when the crystal structures of OmpF and PhoE were solved in 1992 (Cowan et al., 1992). Various structural aspects of the OmpF porin are illustrated in Figure 2. Each monomer is a barrel of 16 b-strands connected by long extracellular loops and short periplasmic turns (Figure 2C). The stability of the trimeric assembly is provided by extensive polypeptide chain contacts between the three monomers, and by the so-called ‘‘latching’’ loop L2 (highlighted in darker color in Figure 2A). This loop emerges from each monomer and extends sideways, crossing over the barrel wall of the adjacent subunit and making ionic contacts with barrel residues of this adjacent subunit. The importance of this loop for the stability of the protein trimer has been demonstrated experimentally (Phale et al., 1998). The b-barrel of each monomer forms a hollow, hydrophilic cylinder that is constricted at mid-height by the inwardly folded L3 loop. This region, the constriction zone or eyelet, is decorated with charged residues that fall into two groups: negatively charged residues on L3 (residues D113 and E117 on OmpF in Figure 2B) and a cluster of positive charges on the opposite barrel wall (residues K16, R42, R82 and R132 on OmpF in Figure 2B). This charge constellation establishes an intrinsic electrostatic potential within the pore itself, that may play an important role in driving ionic movement and in ionic selectivity, as variations in the potential profile have been found between cationand anion-selective porins (Karshikoff et al., 1994; Zeth et al., 2000). These porins were the first ion channel types for which a three-dimensional structure was available, and this finding paved the way for numerous investigations of structure-function relationships (Saint et al., 1996a; Eppens et al., 1997; Phale et al., 1997, 1998; Van Gelder et al., 1997; Bainbridge et al., 1998; *For correspondence. Email. [email protected]; Tel. (713) 743-2684; Fax. (713) 743-2636. J. Mol. Microbiol. Biotechnol. (2002) 4(1): 1–10. JMMB Symposium # 2002 Horizon Scientific Press Liu and Delcour, 1998a; Liu et al., 2000). Most of these studies combined site-directed mutagenesis with electrophysiology, either by measurements on planar lipid bilayers (also known as black lipid membranes or BLM’s) or by patch clamp (see Delcour, 1997 for a comparison of the two techniques). The planar lipid bilayer studies established the importance of the constriction zone for permeation and ionic selectivity (Bauer et al., 1989; Lakey et al., 1991; Saint et al., 1996a). They also shed some light on the role of charged residues of the eyelet in voltage sensing. It has been long known that membrane potentials greater than a certain threshold trigger porin inactivation, leading to a substantial decrease in the current passing through a porin-containing membrane. The mutation of negatively charged residues of L3 into neutral ones was shown to render OmpF less voltagesensitive, but PhoE more voltage-sensitive (Van Gelder et al., 1997). On the other hand, increased voltage-sensitivity was conferred to OmpF by the removal of charges from the cluster of positively charged amino acids located in the barrel wall directly opposite to L3 (Saint et al., 1996a). This intriguing observation led to the suggestion that the positive residues in the barrel are the voltage sensors in the anion-selective PhoE, while the negative charges on the L3 loop are responsible for voltage sensing in the cation-selective porins, such as OmpF and OmpC (Van Gelder et al., 1997). This difference between the two porin types was later correlated to their opposite voltage-dependence (Samartzidou and Delcour, 1998). The position of the L3 loop across the pore makes it a prime candidate for the role of a gate that might swing across the pore to bring about porin inactivation and closure. Recent experiments strongly disputed a gross movement of the L3 loop during voltage-dependent gating, because no change in voltage-sensitivity is observed when the loop is tethered to the barrel by engineered disulfide bridges (Eppens et al., 1997; Phale et al., 1997; Bainbridge et al., 1998). But there remains the possibility that more subtle or localized movements, or a breathing of the two branches of the L3 loop with respect to each other, take place during the putative conformational change underlying voltage-dependent porin inactivation. Black lipid membranes studies on porins have tended to focus on conductance measurements or macroscopic properties of populations of channels. Because higher filter corner frequency and faster sampling are typically used in patch-clamp studies, rapid small transitions in current values, usually masked in traces obtained from BLMs, are detected with the patch clamp technique (Berrier et al., 1992, 1997; Delcour, 1997; Liu and Delcour, 1998a; Liu et al., 2000). These fast transitions between closed and open states suggest that the porins can undergo a spontaneous form of gating, that is distinct from the voltage-induced inactivation. Although one might be Table: Bacterial b-barrel transport proteins with solved oligomeric crystal structures at high resolution. Low-resolution X-ray structures, models, AFM and electron microscopy images are not listed. Protein Bacterium Function Reference Porin Rhodobacter capsulatus General diffusion porin Weiss et al. (1991) Porin Rhodopseudomonas blastica General diffusion porin Kreusch et al. (1994) OmpF Escherichia coli General diffusion porin Cowan et al. (1992) PhoE Escherichia coli General diffusion porin Cowan et al. (1992) OmpK36 Klebsiella pneumoniae General diffusion porin Dutzler et al. (1999) Omp32 Comamonas acidovorans General diffusion porin Zeth et al. (2000) LamB Escherichia coli Specific porin Schirmer et al. (1995) ScrY Salmonella typhimurium Specific porin Forst et al. (1998) FepA Escherichia coli Ligand-gated porin Buchanan et al. (1999) FhuA Escherichia coli Ligand-gated porin Fergusson et al. (1998) Locher et al. (1998) TolC Escherichia coli Multidrug efflux system Koronakis et al. (2000) a-Hemolysin Staphylococcus aureus Pore toxin Song et al. (1996) Figure 1. X-ray structures of three representative members of the b-barrel families described in the text. Extracellular side is at the top. The structures were drawn with Rasmol. 2 Delcour