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Abstract Background Achieving a high response rate in a physician survey is challenging. Monetary incentives increase response rates but obviously add cost to a survey project. We wondered whether an offer of a free continuing medical education (CME) activity would be effective in improving survey response rate. Results As part of a survey of a national sample of physicians, we randomized half to an offer for a free on-line CME activity upon completion of a web-based survey and the other half to no such offer. We compared response rates between the groups. A total of 1214 out of 8477 potentially eligible physicians responded to our survey, for an overall response rate of 14.3%. The response rate among the control group (no offer of CME credit) was 16.6%, while among those offered the CME opportunity, the response rate was 12.0% (p < 0.0001). Conclusions An offer for a free on-line CME activity did not improve physician survey response rate. On the contrary, the offer for a free CME activity actually appeared to worsen the response rate.

Abstract Background Global coronary heart disease (CHD) risk assessment is recommended to guide primary preventive pharmacotherapy. However, little is known about physicians' understanding and use of global CHD risk assessment. Our objective was to examine US physicians' awareness, use, and attitudes regarding global CHD risk assessment in clinical practice, and how these vary by provider specialty. Methods Using a web-based survey of US family physicians, general internists, and cardiologists, we examined awareness of tools available to calculate CHD risk, method and use of CHD risk assessment, attitudes towards CHD risk assessment, and frequency of using CHD risk assessment to guide recommendations of aspirin, lipid-lowering and blood pressure (BP) lowering therapies for primary prevention. Characteristics of physicians indicating they use CHD risk assessments were compared in unadjusted and adjusted analyses. Results A total of 952 physicians completed the questionnaire, with 92% reporting awareness of tools available to calculate CHD global risk. Among those aware of such tools, over 80% agreed that CHD risk calculation is useful, improves patient care, and leads to better decisions about recommending preventive therapies. However, only 41% use CHD risk assessment in practice. The most commonly reported barrier to CHD risk assessment is that it is too time consuming. Among respondents who calculate global CHD risk, 69% indicated they use it to guide lipid lowering therapy recommendations; 54% use it to guide aspirin therapy recommendations; and 48% use it to guide BP lowering therapy. Only 40% of respondents who use global CHD risk routinely tell patients their risk. Use of a personal digital assistant or smart phone was associated with reported use of CHD risk assessment (adjusted OR 1.58; 95% CI 1.17-2.12). Conclusions Reported awareness of tools to calculate global CHD risk appears high, but the majority of physicians in this sample do not use CHD risk assessments in practice. A minority of physicians in this sample use global CHD risk to guide prescription decisions or to motivate patients. Educational interventions and system improvements to improve physicians' effective use of global CHD risk assessment should be developed and tested.

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P. vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of a PvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials., (open access.)

GPR65 is a proton-sensing G-protein coupled receptor associated with multiple immune-mediated inflammatory diseases, whose function is relatively poorly understood. With few reagents commercially available to probe the biology of receptor, generation of an anti-GPR65 monoclonal antibody was desired. Using soluble chimeric scaffolds, such as ApoE3, displaying the extracellular loops of GPR65, together with established phage display technology, native GPR65 loop-specific antibodies were identified. Phage-derived loop-binding antibodies recognized the wild-type native receptor to which they had not previously been exposed, generating confidence in the use of chimeric soluble proteins to act as efficient surrogates for membrane protein extracellular loop antigens. This technique provides promise for the rational design of chimeric antigens in facilitating the discovery of specific antibodies to GPCRs.

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine., Competing Interests: Declaration of interests Ted Ross is an inventor on influenza vaccines., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Low blood concentrations of glucose (hypoglycaemia) soon after birth are common because of the delayed metabolic transition from maternal to endogenous neonatal sources of glucose. Because glucose is the main energy source for the brain, severe hypoglycaemia can cause neuroglycopenia (inadequate supply of glucose to the brain) and, if severe, permanent brain injury. Routine screening of infants at risk and treatment when hypoglycaemia is detected are therefore widely recommended. Robust evidence to support most aspects of management is lacking, however, including the appropriate threshold for diagnosis and optimal monitoring. Treatment is usually initially more feeding, with buccal dextrose gel, followed by intravenous dextrose. In infants at risk, developmental outcomes after mild hypoglycaemia seem to be worse than in those who do not develop hypoglycaemia, but the reasons for these observations are uncertain. Here, the current understanding of the pathophysiology of neonatal hypoglycaemia and recent evidence regarding its diagnosis, management, and outcomes are reviewed. Recommendations are made for further research priorities., Competing Interests: Competing interests: We have read and understood the BMJ policy on declaration of interests and declare the following interests: none., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The rise in antimicrobial resistance is a global health crisis and necessitates the development of novel strategies to treat infections. For example, in 2022 tuberculosis (TB) was the second leading infectious killer after COVID-19, with multi-drug-resistant strains of TB having an ∼40% fatality rate. Targeting essential biosynthetic pathways in pathogens has proven to be successful for the development of novel antimicrobial treatments. Fatty-acid synthesis (FAS) in bacteria proceeds via the type II pathway, which is substantially different from the type I pathway utilized in animals. This makes bacterial fatty-acid biosynthesis (Fab) enzymes appealing as drug targets. FabG is an essential FASII enzyme, and some bacteria, such as Mycobacterium tuberculosis, the causative agent of TB, harbor multiple homologs. FabG4 is a conserved, high-molecular-weight FabG (HMwFabG) that was first identified in M. tuberculosis and is distinct from the canonical low-molecular-weight FabG. Here, structural and functional analyses of Mycolicibacterium smegmatis FabG4, the third HMwFabG studied to date, are reported. Crystal structures of NAD + and apo MsFabG4, along with kinetic analyses, show that MsFabG4 preferentially binds and uses NADH when reducing CoA substrates. As M. smegmatis is often used as a model organism for M. tuberculosis, these studies may aid the development of drugs to treat TB and add to the growing body of research that distinguish HMwFabGs from the archetypal low-molecular-weight FabG.

Background: Clinical trials showed a single oral dose of doxycycline taken after sex protects against STIs among men who have sex with men (MSM) but not women. Pharmacokinetic data at vaginal, rectal and penile sites of STI exposure are lacking. We examined vaginal, rectal and urethral doxycycline concentrations in men and women to better inform STI prevention., Methods: Doxycycline pharmacokinetics in male and female participants 18-59 years of age were evaluated in blood and urine and on rectal and vaginal swabs collected at 1, 2, 4, 8, 24, 48, 72, 96 and 168 h after receiving a 200 mg oral doxycycline dose in a non-randomised single dose open label single centre study in Atlanta, Georgia. Rectal, vaginal, and cervical biopsies and male urethral swabs were collected 24 h after dosing (Trial registration: NCT04860505). Doxycycline was measured by liquid chromatography-mass spectrometry., Findings: Eleven male and nine female participants participated in the study. Doxycycline concentrations on rectal and vaginal swabs collected up to 96 h after dosing were approximately twice those of plasma and remained above minimum inhibitory concentrations (MICs) for at least four, three, and two days for Chlamydia trachomatis, Treponema pallidum, and tetracycline-sensitive Neisseria gonorrhoeae, respectively. Geometric mean doxycycline concentrations in male urethral secretions (1.166 μg/mL; 95% CI 0.568-2.394 μg/mL), male rectal (0.596 μg/g; 0.442-0.803 μg/g), vaginal (0.261 μg/g; 0.098-0.696 μg/g) and cervical tissue (0.410 μg/g; 0.193-0.870 μg/g) in biopsies collected 24 h after dosing exceeded MICs. Plasma and urine doxycycline levels defined adherence markers up to four and seven days postdosing, respectively. No adverse events were reported in this study., Interpretation: Doxycycline efficiently distributes to the rectum, vagina and urethra. Findings can help explain efficacy of STI prevention by doxycycline., Funding: Funded by CDC intramural funds, CDC contract HCVJCG-2020-45044 (to CFK)., Competing Interests: Declaration of interests C.F.K. reports grants to her institution from Moderna, Novavax, Gilead, ViiV and Humanigen for work not related to this manuscript. All other authors have no competing interests to declare. The findings and conclusions in this manuscript are those of the authors and do not necessarily represent the official position of the United States Centers for Disease Control and Prevention or the Department of Health and Human Services., (Published by Elsevier B.V.)

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13-19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate T m values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.

Inorganic pyrophosphate (PP i ) is generated as an intermediate or byproduct of many fundamental metabolic pathways, including DNA/RNA synthesis. The intracellular concentration of PP i must be regulated as buildup can inhibit many critical cellular processes. Inorganic pyrophosphatases (PPases) hydrolyze PP i into two orthophosphates (P i ), preventing the toxic accumulation of the PP i byproduct in cells and making P i available for use in biosynthetic pathways. Here, the crystal structure of a family I inorganic pyrophosphatase from Legionella pneumophila is reported at 2.0 Å resolution. L. pneumophila PPase (LpPPase) adopts a homohexameric assembly and shares the oligonucleotide/oligosaccharide-binding (OB) β-barrel core fold common to many other bacterial family I PPases. LpPPase demonstrated hydrolytic activity against a general substrate, with Mg 2+ being the preferred metal cofactor for catalysis. Legionnaires' disease is a severe respiratory infection caused primarily by L. pneumophila, and thus increased characterization of the L. pneumophila proteome is of interest.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N 5 -CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N 5 -CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC 50 values. Several of these compounds, including the α 1 -blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

Introduction: Treatment options against Mycobacterium abscessus infections are very limited. New compounds are needed to cure M. abscessus pulmonary diseases. While the mycolic acid biosynthetic pathway has been largely exploited for the treatment of tuberculosis, this metabolic process has been overlooked in M. abscessus , although it offers many potential drug targets for the treatment of this opportunistic pathogen., Areas Covered: Herein, the authors review the role of the MmpL3 membrane protein and the enoyl-ACP reductase InhA involved in the transport and synthesis of mycolic acids, respectively. They discuss their importance as two major vulnerable drug targets in M. abscessus and report the activity of MmpL3 and InhA inhibitors. In particular, they focus on NITD-916, a direct InhA inhibitor against M. abscessus , particularly warranted in the context of multidrug resistance., Expert Opinion: There is an increasing body of evidence validating the mycolic acid pathway as an attractive drug target to be further exploited for M. abscessus lung disease treatments. The NITD-916 studies provide a proof-of-concept that direct inhibitors of InhA are efficient in vitro, in macrophages and in zebrafish. Future work is now required to improve the activity and pharmacological properties of these inhibitors and their evaluation in pre-clinical models.

Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines., (© 2023. The Author(s).)

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhA MFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhA MFO . Importantly, single nucleotide polymorphism leading to a G96S substitution in InhA MFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.

Background: Event-driven HIV prevention strategies are a priority for users who do not require daily pre-exposure prophylaxis (PrEP). Regimens containing integrase strand transfer inhibitors (INSTIs) are under evaluation as alternatives to daily PrEP. To better understand INSTI distribution and inform dosing selection we compared the pharmacology of two-dose boosted elvitegravir and unboosted bictegravir regimens in MSM., Materials and Methods: Blood, rectal and penile secretions and rectal biopsies were collected from 63 HIV-negative MSM aged 18-49 years. Specimens were collected up to 96 h after two oral doses of tenofovir alafenamide and emtricitabine with elvitegravir boosted by cobicistat or unboosted bictegravir given 24 h apart. Antiretroviral drugs were measured by LC-MS., Results: Mean bictegravir plasma concentrations remained above the 95% protein-adjusted effective concentration 96 h after dosing [273 (95% CI: 164-456) ng/mL] whereas elvitegravir plasma concentrations became undetectable 48 h after the second dose. Bictegravir and elvitegravir reached rectal tissues within 2 h after the first dose, and elvitegravir tissue concentrations [1.07 (0.38-13.51) ng/mg] were greater than bictegravir concentrations [0.27 (0.15-0.70) ng/mg]. Both INSTIs became undetectable in tissues within 96 h. Elvitegravir and bictegravir were not consistently detected in penile secretions., Conclusions: Whereas bictegravir plasma concentrations persist at least 4 days after a two-oral-dose HIV prophylaxis regimen, elvitegravir accumulates in mucosal tissues. Differing elvitegravir and bictegravir distribution may result in variable mucosal and systemic antiviral activity and can inform dosing strategies for event-driven HIV prevention., (Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy 2022.)

There is an unmet medical need for effective treatments against Mycobacterium abscessus pulmonary infections, to which cystic fibrosis (CF) patients are particularly vulnerable. Recent studies showed that the antitubercular drug isoniazid is inactive against M. abscessus due to the incapacity of the catalase-peroxidase to convert the pro-drug into a reactive metabolite that inhibits the enoyl-ACP reductase InhA. To validate InhA MAB as a druggable target in M. abscessus , we assayed the activity of NITD-916, a 4-hydroxy-2-pyridone lead candidate initially described as a direct inhibitor of InhA that bypasses KatG bioactivation in Mycobacterium tuberculosis . The compound displayed low MIC values against rough and smooth clinical isolates in vitro and significantly reduced the bacterial burden inside human macrophages. Moreover, treatment with NITD-916 reduced the number and size of intracellular mycobacterial cords, regarded as markers of the severity of the infection. Importantly, NITD-916 significantly lowered the M. abscessus burden in CF-derived lung airway organoids. From a mechanistic perspective, NITD-916 abrogated de novo synthesis of mycolic acids and NITD-916-resistant spontaneous mutants harbored point mutations in InhA MAB at residue 96. That NITD-916 targets InhA MAB directly without activation requirements was confirmed genetically and by resolving the crystal structure of the protein in complex with NADH and NITD-916. These findings collectively indicate that InhA MAB is an attractive target to be exploited for future chemotherapeutic developments against this difficult-to-treat mycobacterium and highlight the potential of NITD-916 derivatives for further evaluation in preclinical settings.

UDP-3- O -( R -3-hydroxymyristoyl)- N -acetylglucosamine deacetylase (LpxC) is a promising drug target in Gram-negative bacteria. Previously, we described a correlation between the residence time of inhibitors on Pseudomonas aeruginosa LpxC ( pa LpxC) and the post-antibiotic effect (PAE) caused by the inhibitors on the growth of P. aeruginosa . Given that drugs with prolonged activity following compound removal may have advantages in dosing regimens, we have explored the structure-kinetic relationship for pa LpxC inhibition by analogues of the pyridone methylsulfone PF5081090 ( 1 ) originally developed by Pfizer. Several analogues have longer residence times on pa LpxC than 1 (41 min) including PT913 , which has a residence time of 124 min. PT913 also has a PAE of 4 h, extending the original correlation observed between residence time and PAE. Collectively, the studies provide a platform for the rational modulation of pa LpxC inhibitor residence time and the potential development of antibacterial agents that cause prolonged suppression of bacterial growth.

E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-electron microscopy (EM) and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E-cadherin monomers, but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent., (© 2022. The Author(s).)

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped β-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites., (© 2022 The Protein Society.)

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections globally and is one of the most commonly reported infections in the United States. There is a need to develop new therapeutics due to drug resistance and the failure of current treatments to clear persistent infections. Structures of potential C. trachomatis rational drug-discovery targets, including C. trachomatis inorganic pyrophosphatase (CtPPase), have been determined by the Seattle Structural Genomics Center for Infectious Disease. Inorganic pyrophosphatase hydrolyzes inorganic pyrophosphate during metabolism. Furthermore, bacterial inorganic pyrophosphatases have shown promise for therapeutic discovery. Here, a 2.2 Å resolution X-ray structure of CtPPase is reported. The crystal structure of CtPPase reveals shared structural features that may facilitate the repurposing of inhibitors identified for bacterial inorganic pyrophosphatases as starting points for new therapeutics for C. trachomatis., (open access.)

Burkholderia pseudomallei infection causes melioidosis, which is often fatal if untreated. There is a need to develop new and more effective treatments for melioidosis. This study reports apo and cofactor-bound crystal structures of the potential drug target betaine aldehyde dehydrogenase (BADH) from B. pseudomallei. A structural comparison identified similarities to BADH from Pseudomonas aeruginosa which is inhibited by the drug disulfiram. This preliminary analysis could facilitate drug-repurposing studies for B. pseudomallei., (open access.)

Members of the bacterial genus Brucella cause brucellosis, a zoonotic disease that affects both livestock and wildlife. Brucella are category B infectious agents that can be aerosolized for biological warfare. As part of the structural genomics studies at the Seattle Structural Genomics Center for Infectious Disease (SSGCID), FolM alternative dihydrofolate reductases 1 from Brucella suis and Brucella canis were produced and their structures are reported. The enzymes share ∼95% sequence identity but have less than 33% sequence identity to other homologues with known structure. The structures are prototypical NADPH-dependent short-chain reductases that share their highest tertiary-structural similarity with protozoan pteridine reductases, which are being investigated for rational therapeutic development., (open access.)

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs., Competing Interests: The authors have declared that no competing interests exist.

Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure-function analyses of the highly versatile FIC domains.

Elizabethkingia anophelis is an emerging multidrug resistant pathogen that has caused several global outbreaks. E. anophelis belongs to the large family of Flavobacteriaceae, which contains many bacteria that are plant, bird, fish, and human pathogens. Several antibiotic resistance genes are found within the E. anophelis genome, including a chloramphenicol acetyltransferase (CAT). CATs play important roles in antibiotic resistance and can be transferred in genetic mobile elements. They catalyse the acetylation of the antibiotic chloramphenicol, thereby reducing its effectiveness as a viable drug for therapy. Here, we determined the high-resolution crystal structure of a CAT protein from the E. anophelis NUHP1 strain that caused a Singaporean outbreak. Its structure does not resemble that of the classical Type A CATs but rather exhibits significant similarity to other previously characterized Type B (CatB) proteins from Pseudomonas aeruginosa, Vibrio cholerae and Vibrio vulnificus, which adopt a hexapeptide repeat fold. Moreover, the CAT protein from E. anophelis displayed high sequence similarity to other clinically validated chloramphenicol resistance genes, indicating it may also play a role in resistance to this antibiotic. Our work expands the very limited structural and functional coverage of proteins from Flavobacteriaceae pathogens which are becoming increasingly more problematic.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen., Competing Interests: The authors have read the journal’s policy and have the following competing interests: DMD, JA, SLD, DRD, SJM, BC, MJB, DDL, and TEE are employees of UCB Pharma. The LS-CAT Sector 21 was supported by a grant from the Michigan Economic Development Corporation. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine β-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure-activity relationship studies.

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ 1 -pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD + and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M -1 s -1 . The reaction is reversible, and nuclear magnetic resonance characterization ( 1 H, 13 C, and 31 P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD + . We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP + -TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P) + . Because NAD(P) + -dependent oxidoreductases are widespread in nature, our results may be broadly relevant.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity., (© 2020 The Protein Society.)

A protein superfamily with a "Domain of Unknown Function,", DUF3349 (PF11829), is present predominately in Mycobacterium and Rhodococcus bacterial species suggesting that these proteins may have a biological function unique to these bacteria. We previously reported the inaugural structure of a DUF3349 superfamily member, Mycobacterium tuberculosis Rv0543c. Here, we report the structures determined for three additional DUF3349 proteins: Mycobacterium smegmatis MSMEG&#95;1063 and MSMEG&#95;1066 and Mycobacterium abscessus MAB&#95;3403c. Like Rv0543c, the NMR solution structure of MSMEG&#95;1063 revealed a monomeric five α-helix bundle with a similar overall topology. Conversely, the crystal structure of MSMEG&#95;1066 revealed a five α-helix protein with a strikingly different topology and a tetrameric quaternary structure that was confirmed by size exclusion chromatography. The NMR solution structure of a fourth member of the DUF3349 superfamily, MAB&#95;3403c, with 18 residues missing at the N-terminus, revealed a monomeric α-helical protein with a folding topology similar to the three C-terminal helices in the protomer of the MSMEG&#95;1066 tetramer. These structures, together with a GREMLIN-based bioinformatics analysis of the DUF3349 primary amino acid sequences, suggest two subfamilies within the DUF3349 family. The division of the DUF3349 into two distinct subfamilies would have been lost if structure solution had stopped with the first structure in the DUF3349 family, highlighting the insights generated by solving multiple structures within a protein superfamily. Future studies will determine if the structural diversity at the tertiary and quaternary levels in the DUF3349 protein superfamily have functional roles in Mycobacteria and Rhodococcus species with potential implications for structure-based drug discovery., (© 2019 The Protein Society.)

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii., (© 2020 The Protein Society.)

Background: Higher-order structure (HOS) assessment is an important component of biosimilarity evaluations. While established spectroscopic methods are routinely used to characterize structure and evaluate similarity, the addition of X-ray crystallographic analysis to these biophysical methods enables orthogonal elucidation of HOS at higher resolution., Methods: Crystal structures of the infliximab biosimilar PF-06438179/GP1111 and the reference product Remicade ® , sourced from US and European Union markets, were determined and compared to evaluate HOS similarity. Analytical ultracentrifugation studies were conducted to understand reversible self-association., Results: In contrast to more routine spectroscopic methods, the crystal structures enable three-dimensional assessment of complementarity-determining regions and other local regions at near-atomic resolution. The biosimilar structures are highly similar to those of the reference product, as demonstrated visually and though all-atom root-mean-squared deviation measurements., Conclusion: The structures provide new insights into the physicochemical properties of the proposed biosimilar and the reference product, further strengthening the 'totality of evidence' in the evaluation of similarity.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase ( Pf KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both Pf KRS1 and C. parvum KRS ( Cp KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED 90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between Pf KRS1 and Cp KRS. This series of compounds inhibit Cp KRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for Pf KRS1 and Cp KRS vs. (human) Hs KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis., Competing Interests: Conflict of interest statement: A patent relating to this work has been filed (PCT/GB2017/051809). F.-J.G. and L.M.S. are employees of GlaxoSmithKline and own shares of the company. M.B.J.-D. and I.A.-B. have shares in The Art of Discovery. Editor D.E.G. is a recent coauthor with two authors of this paper. He published a research article with M.A. in 2015. With E.A.W. he published two research articles in 2016, one research article in 2018, and coauthored a research article forthcoming in 2019. D.E.G. is a coinvestigator with E.A.W. on a 2012–2019 grant., (Copyright © 2019 the Author(s). Published by PNAS.)

Thiamine monophosphate kinase (ThiL) catalyzes the last step of thiamine pyrophosphate (TPP) synthesis, the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to thiamine pyrophosphate. We solved the structure of ThiL from the human pathogen A. baumanii in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate. High resolution of the data and anomalous diffraction allows for a detailed description of the binding mode of substrates and products, and their metal environment. The structures further support a previously proposed in-line attack reaction mechanism and show a distinct variability of metal content of the active site.

There are several options available for intravenous application of iron supplements, but they all have a similar structure:-an iron core surrounded by a carbohydrate coating. These nanoparticles require processing by the reticuloendothelial system to release iron, which is subsequently picked up by the iron-binding protein transferrin and distributed throughout the body, with most of the iron supplied to the bone marrow. This process risks exposing cells and tissues to free iron, which is potentially toxic due to its high redox activity. A new parenteral iron formation, ferric pyrophosphate citrate (FPC), has a novel structure that differs from conventional intravenous iron formulations, consisting of an iron atom complexed to one pyrophosphate and two citrate anions. In this study, we show that FPC can directly transfer iron to apo-transferrin. Kinetic analyses reveal that FPC donates iron to apo-transferrin with fast binding kinetics. In addition, the crystal structure of transferrin bound to FPC shows that FPC can donate iron to both iron-binding sites found within the transferrin structure. Examination of the iron-binding sites demonstrates that the iron atoms in both sites are fully encapsulated, forming bonds with amino acid side chains in the protein as well as pyrophosphate and carbonate anions. Taken together, these data demonstrate that, unlike intravenous iron formulations, FPC can directly and rapidly donate iron to transferrin in a manner that does not expose cells and tissues to the damaging effects of free, redox-active iron.

Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2 Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8 kDa protein sample and yielded a 1.6 Å resolution data set with a unit cell that could accommodate approximately 8 kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.

Burkholderia thailandensis is often used as a model for more virulent members of this genus of proteobacteria that are highly antibiotic-resistant and are potential agents of biological warfare that are infective by inhalation. As part of ongoing efforts to identify potential targets for the development of rational therapeutics, the structures of enzymes that are absent in humans, including that of chorismate mutase from B. thailandensis, have been determined by the Seattle Structural Genomics Center for Infectious Disease. The high-resolution structure of chorismate mutase from B. thailandensis was determined in the monoclinic space group P2 1 with three homodimers per asymmetric unit. The overall structure of each protomer has the prototypical AroQγ topology and shares conserved binding-cavity residues with other chorismate mutases, including those with which it has no appreciable sequence identity.

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose-response curves and 11 compounds were found to inhibit enzymatic activity with IC 50 values of less than 10 µM. Finally, compounds (1-5) were docked against RpMetAP with AutoDock to determine potential binding mechanisms and the results were compared with crystal structures deposited within the PDB., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The bacterium Burkholderia phymatum is a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. Other Burkholderia species are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase from B. phymatum was produced and crystallized, and a 1.95 Å resolution structure is reported. This enzyme shares less than 33% sequence identity with other homologs of known structure. There are two classes of chorismate mutase: AroQ and AroH. The bacterial subclass AroQγ has reported roles in virulence. Chorismate mutase from B. phymatum has the prototypical AroQγ topology and retains the characteristic chorismate mutase active site. This suggests that substrate-based chorismate mutase inhibitors will not be specific and are likely to affect beneficial bacteria such as B. phymatum.

The genome of the human pathogen Mycobacterium tuberculosis (Mtb) encodes ∼4,400 proteins, but one third of them have unknown functions. We solved the crystal structure of Rv3651, a hypothetical protein with no discernible similarity to proteins with known function. Rv3651 has a three-domain architecture that combines one cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA (GAF) domain and two Per-ARNT-Sim (PAS) domains. GAF and PAS domains are sensor domains that are typically linked to signaling effector molecules. Unlike these sensor-effector proteins, Rv3651 is an unusual sensor domain-only protein with highly divergent sequence. The structure suggests that Rv3651 integrates multiple different signals and serves as a scaffold to facilitate signal transfer., (© 2017 The Protein Society.)

Background: Relative telomere length in peripheral blood leukocytes has been evaluated as a potential biomarker for renal cell carcinoma (RCC) risk in several studies, with conflicting findings., Objective: We performed an analysis of genetic variants associated with leukocyte telomere length to assess the relationship between telomere length and RCC risk using Mendelian randomization, an approach unaffected by biases from temporal variability and reverse causation that might have affected earlier investigations., Design, Setting, and Participants: Genotypes from nine telomere length-associated variants for 10 784 cases and 20 406 cancer-free controls from six genome-wide association studies (GWAS) of RCC were aggregated into a weighted genetic risk score (GRS) predictive of leukocyte telomere length., Outcome Measurements and Statistical Analysis: Odds ratios (ORs) relating the GRS and RCC risk were computed in individual GWAS datasets and combined by meta-analysis., Results and Limitations: Longer genetically inferred telomere length was associated with an increased risk of RCC (OR=2.07 per predicted kilobase increase, 95% confidence interval [CI]:=1.70-2.53, p<0.0001). As a sensitivity analysis, we excluded two telomere length variants in linkage disequilibrium (R 2 >0.5) with GWAS-identified RCC risk variants (rs10936599 and rs9420907) from the telomere length GRS; despite this exclusion, a statistically significant association between the GRS and RCC risk persisted (OR=1.73, 95% CI=1.36-2.21, p<0.0001). Exploratory analyses for individual histologic subtypes suggested comparable associations with the telomere length GRS for clear cell (N=5573, OR=1.93, 95% CI=1.50-2.49, p<0.0001), papillary (N=573, OR=1.96, 95% CI=1.01-3.81, p=0.046), and chromophobe RCC (N=203, OR=2.37, 95% CI=0.78-7.17, p=0.13)., Conclusions: Our investigation adds to the growing body of evidence indicating some aspect of longer telomere length is important for RCC risk., Patient Summary: Telomeres are segments of DNA at chromosome ends that maintain chromosomal stability. Our study investigated the relationship between genetic variants associated with telomere length and renal cell carcinoma risk. We found evidence suggesting individuals with inherited predisposition to longer telomere length are at increased risk of developing renal cell carcinoma., (Published by Elsevier B.V.)

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn-DX-2507 Fab complex, revealing a nearly complete overlap of the IgG-Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aggregation and self-association in protein-based biotherapeutics are critical quality attributes that are tightly controlled by the manufacturing process. Aggregates have the potential to elicit immune reactions, including neutralizing anti-drug antibodies, which can diminish the drug's efficacy upon subsequent dosing. The structural basis of reversible self-association, a form of non-covalent aggregation in the native state, is only beginning to emerge for many biologics and is often unique to a given molecule. In the present study, crystal structures of the infliximab (Remicade) Fc and Fab domains were determined. The Fab domain structures are the first to be reported in the absence of the antigen (i.e., tumor necrosis factor), and are consistent with a mostly rigid complementarity-determining region loop structure and rotational flexibility between variable and constant regions. A potential self-association interface is conserved in two distinct crystal forms of the Fab domain, and solution studies further demonstrate that reversible self-association of infliximab is mediated by the Fab domain. The crystal structures and corresponding solution studies help rationalize the propensity for infliximab to self-associate and provide insights for the design of improved control strategies in biotherapeutics development.