Bacterial multidrug efflux pumps: structure, function and regulation

The emergence and spread of multidrug resistance among human pathogenic bacteria is an increasing worldwide problem. A comprehensive understanding of the molecular basis of resistance mechanisms of bacteria will be vital for the future development of new and more effective antibiotics and for novel therapeutic treatment strategies. One of the common resistance mechanisms is the active efflux of toxic compounds from the cell by bacterial multidrug efflux systems, which are polyspecific and able to accommodate a variety of structurally and functionally unrelated compounds. Moreover, it is well recognized that the expression of these multidrug efflux transporters is tightly controlled by transcriptional regulators with the same multidrug recognition properties. Elucidation of structural and functional relationships of these multidrug efflux transporters and transcriptional regulators is the major concern of this dissertation. In order to understand the role of Rv3066 in regulating the expression level of the multidrug efflux pump Mmr in M. tuberculosis, we have determined the crystal structure of Rv3066, both in the absence and presence of ethidium bromide. With the aid of other experimental data, induction mechanism of Rv3066 is studied in detail. Currently available various methodologies to crystallize membrane proteins have also been outlined in this dissertation. Towards understanding the molecular mechanism of MtrCDE efflux pump of N. gonorrhoeae, which mediates the export of several structurally diverse toxic chemicals, we have determined the crystal structure of MtrD. In addition, the Mtr efflux system includes another inner membrane protein MtrF, which belongs to the AbgT family of transporters. To date, approximately 13,000 putative transporters of this family have been identified. However, no structural information has been made available and even functional data are fairly minimal. To understand how members of the AbgT family function, we have determined the crystal structure of MtrF and combination of functional studies suggests that MtrF acts as an antibiotic efflux pump. In addition, we have exploited our knowledge on AbgT transporter family, by determining the crystal structure of another member YdaH, of this family. Based on our observations, we believe that many members of the AbgT family transporters may serve as antimetabolite efflux pumps to protect cells against these noxious agents

The structural basis for CD36 binding by the malaria parasite

CD36 is a scavenger receptor involved in fatty acid metabolism, innate immunity and angiogenesis. It interacts with lipoprotein particles and facilitates uptake of long chain fatty acids. It is also the most common target of the PfEMP1 proteins of the malaria parasite, Plasmodium falciparum, tethering parasite-infected erythrocytes to endothelial receptors. This prevents their destruction by splenic clearance and allows increased parasitaemia. Here we describe the structure of CD36 in complex with long chain fatty acids and a CD36-binding PfEMP1 protein domain. A conserved hydrophobic pocket allows the hugely diverse PfEMP1 protein family to bind to a conserved phenylalanine residue at the membrane distal tip of CD36. This phenylalanine is also required for CD36 to interact with lipoprotein particles. By targeting a site on CD36 that is required for its physiological function, PfEMP1 proteins maintain the ability to tether to the endothelium and avoid splenic clearance

Crystal structure of the Alcanivorax borkumensis YdaH transporter reveals an unusual topology

The potential of the folic acid biosynthesis pathway as a target for the development of antibiotics has been clinically validated. However, many pathogens have developed resistance to these antibiotics, prompting a re-evaluation of potential drug targets within the pathway. The ydaH gene of Alcanivorax borkumensis encodes an integral membrane protein of the AbgT family of transporters for which no structural information was available. Here we report the crystal structure of A. borkumensis YdaH, revealing a dimeric molecule with an architecture distinct from other families of transporters. YdaH is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins that suggest a plausible pathway for substrate transport. Further analyses also suggest that YdaH could act as an antibiotic efflux pump and mediate bacterial resistance to sulfonamide antimetabolite drugs

Structural and functional analysis of the transcriptional regulator Rv3066 of Mycobacterium tuberculosis

The Mmr multidrug efflux pump recognizes and actively extrudes a broad range of antimicrobial agents, and promotes the intrinsic resistance to these antimicrobials in Mycobacterium tuberculosis . The expression of Mmr is controlled by the TetR-like transcriptional regulator Rv3066, whose open reading frame is located downstream of the mmr operon. To understand the structural basis of Rv3066 regulation, we have determined the crystal structures of Rv3066, both in the absence and presence of bound ethidium, revealing an asymmetric homodimeric two-domain molecule with an entirely helical architecture. The structures underscore the flexibility and plasticity of the regulator essential for multidrug recognition. Comparison of the apo-Rv3066 and Rv3066–ethidium crystal structures suggests that the conformational changes leading to drug-mediated derepression is primarily due to a rigid body rotational motion within the dimer interface of the regulator. The Rv3066 regulator creates a multidrug-binding pocket, which contains five aromatic residues. The bound ethidium is found buried within the multidrug-binding site, where extensive aromatic stacking interactions seemingly govern the binding. In vitro studies reveal that the dimeric Rv3066 regulator binds to a 14-bp palindromic inverted repeat sequence in the nanomolar range. These findings provide new insight into the mechanisms of ligand binding and Rv3066 regulation

Crystal Structure of the Open State of the Neisseria gonorrhoeae MtrE Outer Membrane Channel

Active efflux of antimicrobial agents is one of the most important strategies used by bacteria to defend against antimicrobial factors present in their environment. Mediating many cases of antibiotic resistance are transmembrane efflux pumps, composed of one or more proteins. The Neisseria gonorrhoeae MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND) family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here describe the crystal structure of N. gonorrhoeae MtrE, the outer membrane component of the MtrCDE tripartite multidrug efflux system. This trimeric MtrE channel forms a vertical tunnel extending down contiguously from the outer membrane surface to the periplasmic end, indicating that our structure of MtrE depicts an open conformational state of this channel

A mass spectrometry-based approach to distinguish annular and specific lipid binding to membrane proteins

Membrane proteins engage in a variety of contacts with theirsurrounding lipids, but distinguishing between specifically boundlipids, and non-specific annular interactionsis a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the Presenilin Homologue protease are subject to constant exchange with detergent. Bycontrast,detergent-resistantlipids bound at the dimer interface in the Leucine transportershowdecreased koffrates in molecular dynamics simulations.Turning tothe lipid flippase MurJ, we findthat addition of the natural substrate lipid-II results in the formation of a 1:1 protein-lipid complex, where the lipid cannot be displaced by detergentfromthe highly protected active site.In summary, we distinguish annular from non-annular lipids based on their exchange rates in solution. [Abstract copyright: © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Accumulator pricing

Accumulator is a highly path dependant derivative structure that has been introduced as a retail financial product in recent years and becomes very popular in some Asian cities with its speculative nature. Despite its popularity, its pricing formula is not well known especially when there is a barrier structure. When the barrier in an accumulator contract is applied continuously, this paper obtains exact analytic pricing formulae for immediate settlement and for delay settlement. For discrete barrier, we also obtain analytic formulae which can approximate the fair price of an accumulator under both settlement methods. Through Monte Carlo simulation, we show that the approximation is highly satisfactory. With price formulae in close forms, this paper further explains how to price the product fairly to fit into its zero-cost structure. The analytic formulae also help in computing the Greeks of an accumulator which are documented in this paper. An asymmetry can be observed here that when the buyer is suffering a loss, risk characteristics like delta and vega are substantially larger than when the buyer is enjoying a profit. This means that losing buyers will be more vulnerable to price changes and volatility changes than winning buyers. This is consistent with another observation in the paper that the value at risk for the buyer can be several times larger than that of the seller. © 2009 IEEE.published_or_final_versionThe IEEE Symposium on Computational Intelligence for Financial Engineering (CIFEr) 2009, Nashville, TN., 30 March-2 April 2009. In Proceedings of the CIFEr, 2009, p. 72-7

Bacterial multidrug efflux pumps: structure, function and regulation

The emergence and spread of multidrug resistance among human pathogenic bacteria is an increasing worldwide problem. A comprehensive understanding of the molecular basis of resistance mechanisms of bacteria will be vital for the future development of new and more effective antibiotics and for novel therapeutic treatment strategies. One of the common resistance mechanisms is the active efflux of toxic compounds from the cell by bacterial multidrug efflux systems, which are polyspecific and able to accommodate a variety of structurally and functionally unrelated compounds. Moreover, it is well recognized that the expression of these multidrug efflux transporters is tightly controlled by transcriptional regulators with the same multidrug recognition properties. Elucidation of structural and functional relationships of these multidrug efflux transporters and transcriptional regulators is the major concern of this dissertation. In order to understand the role of Rv3066 in regulating the expression level of the multidrug efflux pump Mmr in M. tuberculosis, we have determined the crystal structure of Rv3066, both in the absence and presence of ethidium bromide. With the aid of other experimental data, induction mechanism of Rv3066 is studied in detail. Currently available various methodologies to crystallize membrane proteins have also been outlined in this dissertation. Towards understanding the molecular mechanism of MtrCDE efflux pump of N. gonorrhoeae, which mediates the export of several structurally diverse toxic chemicals, we have determined the crystal structure of MtrD. In addition, the Mtr efflux system includes another inner membrane protein MtrF, which belongs to the AbgT family of transporters. To date, approximately 13,000 putative transporters of this family have been identified. However, no structural information has been made available and even functional data are fairly minimal. To understand how members of the AbgT family function, we have determined the crystal structure of MtrF and combination of functional studies suggests that MtrF acts as an antibiotic efflux pump. In addition, we have exploited our knowledge on AbgT transporter family, by determining the crystal structure of another member YdaH, of this family. Based on our observations, we believe that many members of the AbgT family transporters may serve as antimetabolite efflux pumps to protect cells against these noxious agents.</p

Structural basis for silicic acid uptake by higher plants

Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity