A structural biology approach to the problem of antibiotic resistance in bacteria

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. We demonstrate how these structural studies can directly contribute to unsolved problems in biology, with a focus on the growing problem of antibiotic resistance in bacterial infections. Multi-drug efflux transporters are common and powerful resistance mechanisms that are capable of extruding a number of structurally unrelated antimicrobials, including antibiotics and toxic heavy metal ions, from the bacterial cell. We begin by presenting the crystal structures of the individual pump components of the Escherichia coli Cus system, a paradigm for efflux machinery, and speculate on how these pumps assemble to fight diverse antimicrobials. In Mycobacterium tuberculosis, the cell wall is critical to the virulence and antimicrobial resistance of these pathogens. Recent work shows that the MmpL transporter family contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the M. tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. We demonstrate how the structures of these two proteins enhance understanding of the MmpL family of proteins and to develop new antibacterial tools to fight tuberculosis. Neisseria gonorrhoeae is a Gram-negative human pathogen and the cause of the STD gonorrhea. In N. gonorrhoeae, the MtrCDE multidrug efflux system mediates resistance to diverse antibiotics, nonionic detergents, antibacterial peptides, bile salts, and steroidal hormones. We have developed several techniques to assemble the complete MtrCDE tripartite efflux complex, which we present here. These efforts have culminated in a low-resolution structure of the bipartite MtrCD complex. Finally, we apply our crystallography techniques to the problem of chloroplast cell division. In plants and algae, chloroplast division proceeds by binary fission, involving the coordinated assembly of four rings, both inside and outside the cell. We have determined the first high-resolution crystal structure of the Arabidopsis thaliana cell division protein PARC6. In addition, we obtained the co-crystal structure of PARC6 and PDV1, another protein within this network, revealing the molecular details of the intermembrane space interaction during chloroplast cell division

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

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

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

A structural biology approach to the problem of antibiotic resistance in bacteria

X-ray crystallography remains the most robust method to determine protein structure at the atomic level. We demonstrate how these structural studies can directly contribute to unsolved problems in biology, with a focus on the growing problem of antibiotic resistance in bacterial infections. Multi-drug efflux transporters are common and powerful resistance mechanisms that are capable of extruding a number of structurally unrelated antimicrobials, including antibiotics and toxic heavy metal ions, from the bacterial cell. We begin by presenting the crystal structures of the individual pump components of the Escherichia coli Cus system, a paradigm for efflux machinery, and speculate on how these pumps assemble to fight diverse antimicrobials. In Mycobacterium tuberculosis, the cell wall is critical to the virulence and antimicrobial resistance of these pathogens. Recent work shows that the MmpL transporter family contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the M. tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR family transcriptional regulators Rv3249c and Rv1816. We demonstrate how the structures of these two proteins enhance understanding of the MmpL family of proteins and to develop new antibacterial tools to fight tuberculosis. Neisseria gonorrhoeae is a Gram-negative human pathogen and the cause of the STD gonorrhea. In N. gonorrhoeae, the MtrCDE multidrug efflux system mediates resistance to diverse antibiotics, nonionic detergents, antibacterial peptides, bile salts, and steroidal hormones. We have developed several techniques to assemble the complete MtrCDE tripartite efflux complex, which we present here. These efforts have culminated in a low-resolution structure of the bipartite MtrCD complex. Finally, we apply our crystallography techniques to the problem of chloroplast cell division. In plants and algae, chloroplast division proceeds by binary fission, involving the coordinated assembly of four rings, both inside and outside the cell. We have determined the first high-resolution crystal structure of the Arabidopsis thaliana cell division protein PARC6. In addition, we obtained the co-crystal structure of PARC6 and PDV1, another protein within this network, revealing the molecular details of the intermembrane space interaction during chloroplast cell division.</p

Machine learning prediction of methionine and tryptophan photooxidation susceptibility

Photooxidation of methionine (Met) and tryptophan (Trp) residues is common and includes major degradation pathways that often pose a serious threat to the success of therapeutic proteins. Oxidation impacts all steps of protein production, manufacturing, and shelf life. Prediction of oxidation liability as early as possible in development is important because many more candidate drugs are discovered than can be tested experimentally. Undetected oxidation liabilities necessitate expensive and time-consuming remediation strategies in development and may lead to good drugs reaching patients slowly. Conversely, sites mischaracterized as oxidation liabilities could result in overengineering and lead to good drugs never reaching patients. To our knowledge, no predictive model for photooxidation of Met or Trp is currently available. We applied the random forest machine learning algorithm to in-house liquid chromatography-tandem mass spectrometry (LC-MS/MS) datasets (Met, n = 421; Trp, n = 342) of tryptic therapeutic protein peptides to create computational models for Met and Trp photooxidation. We show that our machine learning models predict Met and Trp photooxidation likelihood with 0.926 and 0.860 area under the curve (AUC), respectively, and Met photooxidation rate with a correlation coefficient (Q2) of 0.511 and root-mean-square error (RMSE) of 10.9%. We further identify important physical, chemical, and formulation parameters that influence photooxidation. Improvement of biopharmaceutical liability predictions will result in better, more stable drugs, increasing development throughput, product quality, and likelihood of clinical success

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.This article is from Nature Communications 6 (2015): 60, doi:10.1038/ncomms7874. Posted with permission.</p

Structure and Function of Neisseria gonorrhoeae MtrF Illuminates a Class of Antimetabolite Efflux Pumps

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF 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, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.This article is from Cell Reports 11 (2015): 61, doi:10.1016/j.celrep.2015.03.003. Posted with permission.</p

Crystal Structure of the Neisseria gonorrhoeae MtrD Inner Membrane Multidrug Efflux Pump

Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The 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 report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps.This article is from PLoS One 9 (2014): 1, doi:10.1371/journal.pone.0097903. Posted with permission.</p