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ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely "regenerated" Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.

Nisin is a posttranslationally modified antimicrobial peptide containing the cyclic thioether amino acids lanthionine and methyllanthionine. Although much is known about its antimicrobial activity and mode of action, knowledge about the nisin modification process is still rather limited. The dehydratase NisB is believed to be the initial interaction partner in modification. NisB dehydrates specific serine and threonine residues in prenisin, whereas the cyclase NisC catalyzes the (methyl) lanthionine formation. The fully modified prenisin is exported and the leader peptide is cleaved off by the extracellular protease NisP. Light scattering analysis demonstrated that purified NisB is a dimer in solution. Using size exclusion chromatography and surface plasmon resonance, the interaction of NisB and prenisin, including several of its modified derivatives, was studied. Unmodified prenisin binds to NisB with an affinity of 1.05 +/- 0.25 mu M, whereas the dehydrated and the fully modified derivatives bind with respective affinities of 0.31 +/- 0.07 and 10.5 +/- 1.7 mu M. The much lower affinity for the fully modified prenisin was related to a > 20-fold higher off-rate. For all three peptides the stoichiometry of binding was 1:1. Active nisin, which is the equivalent of fully modified prenisin lacking the leader peptide did not bind to NisB, nor did prenisin in which the highly conserved FNLD box within the leader peptide was mutated to AAAA. Taken together our data indicate that the leader peptide is essential for initial recognition and binding of prenisin to NisB.

International audience; Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.