Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-ß and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest. In this work, we explored a panel of ubiquitin variants (UbVs) that were previously identified to bind Smurf1 or Smurf2. In vitro binding and ubiquitination assays identified a highly specific Smurf2 inhibitor, UbV S2.4, which was able to inhibit ligase activity with high potency in the low nanomolar range. Orthologous cellular assays further demonstrated high specificity of UbV S2.4 toward Smurf2 and no cross-reactivity toward Smurf1. Structural analysis of UbV S2.4 in complex with Smurf2 revealed its mechanism of inhibition was through targeting the E2 binding site. In summary, we investigated several protein-based inhibitors of Smurf1 and Smurf2 and identified a highly specific Smurf2 inhibitor that disrupts the E2-E3 protein interaction interface.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Engineered SH2 Domains for Targeted Phosphoproteomics.

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes1) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2 superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

Management of cough in patients with idiopathic interstitial lung diseases in primary care.

IMPORTANCE: Cough is a common symptom in idiopathic interstitial lung diseases (ILDs), there is little information of its management in primary care. The objective of this study was to explore the frequency of cough-related consultations and the medications prescribed to patients with ILDs in primary care. METHODS: This retrospective cohort study used electronic medical records (EMR) from Manitoba primary care providers participating in the Manitoba Primary Care Research Network repository (2014-2019). Cough-related consults and the subsequent medications prescribed to patients with ILDs were identified in the EMR. RESULTS: 295 patients with ILDs were identified, 73 (25%) of them had 141 cough-related consultations (mean 1.9, SD 1.3) during the period studied. In 50 (35%) of the consultations, patients were prescribed one or more of the following: inhaled bronchodilators (34%), nasal corticoids (18%), codeine/opiates (18%), antibiotics (14%), inhaled corticoids (14%), proton pump inhibitors (8%), cough preparations (6%), antihistamines (4%), and oral corticoids (2%). 13 (26%) subsequent cough-related consultations were identified within 6 months, mainly among patients who were prescribed cough preparations, nasal corticoids, antihistamines, and antibiotics. CONCLUSION: One-quarter of patients with ILDs consulted primary care due to cough, and about a third of them received a prescription to address potentially underlying causes of cough. Although further studies are required to explore the effect of the medications prescribed, recurrent cough consultations suggested that cough preparations, nasal corticoids, and antihistamines are among the least effective treatments. More research is needed to understand the causes and optimal treatment of cough in patients with ILDs.

Panel of Engineered Ubiquitin Variants Targeting the Family of Human Ubiquitin Interacting Motifs.

Ubiquitin (Ub)-binding domains embedded in intracellular proteins act as readers of the complex Ub code and contribute to regulation of numerous eukaryotic processes. Ub-interacting motifs (UIMs) are short α-helical modular recognition elements whose role in controlling proteostasis and signal transduction has been poorly investigated. Moreover, impaired or aberrant activity of UIM-containing proteins has been implicated in numerous diseases, but targeting modular recognition elements in proteins remains a major challenge. To overcome this limitation, we developed Ub variants (UbVs) that bind to 42 UIMs in the human proteome with high affinity and specificity. Structural analysis of a UbV:UIM complex revealed the molecular determinants of enhanced affinity and specificity. Furthermore, we showed that a UbV targeting a UIM in the cancer-associated Ub-specific protease 28 potently inhibited catalytic activity. Our work demonstrates the versatility of UbVs to target short α-helical Ub receptors with high affinity and specificity. Moreover, the UbVs provide a toolkit to investigate the role of UIMs in regulating and transducing Ub signals and establish a general strategy for the systematic development of probes for Ub-binding domains.

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs).

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.

Comprehensive Assessment of the Relationship Between Site-2 Specificity and Helix α2 in the Erbin PDZ Domain.

PDZ domains are key players in signalling pathways. These modular domains generally recognize short linear C-terminal stretches of sequences in proteins that organize the formation of complex multi-component assemblies. The development of new methodologies for the characterization of the molecular principles governing these interactions is critical to fully understand the functional diversity of the family and to elucidate biological functions for family members. Here, we applied an in vitro evolution strategy to explore comprehensively the capacity of PDZ domains for specific recognition of different amino acids at a key position in C-terminal peptide ligands. We constructed a phage-displayed library of the Erbin PDZ domain by randomizing the binding site-2 and adjacent residues, which are all contained in helix α2, and we selected for variants binding to a panel of peptides representing all possible position-2 residues. This approach generated insights into the basis for the common natural class I and II specificities, demonstrated an alternative basis for a rare natural class III specificity for Asp-2, and revealed a novel specificity for Arg-2 that has not been reported in natural PDZ domains. A structure of a PDZ-peptide complex explained the minimum requirement for switching specificity from class I ligands containing Thr/Ser-2 to class II ligands containing hydrophobic residues at position-2. A second structure explained the molecular basis for the specificity for ligands containing Arg-2. Overall, the evolved PDZ variants greatly expand our understanding of site-2 specificities and the variants themselves may prove useful as building blocks for synthetic biology.

How long are Canadians waiting to access specialty care? Retrospective study from a primary care perspective.

OBJECTIVE: To calculate patient wait times for specialist care using data from primary care clinics across Canada. DESIGN: Retrospective chart audit. SETTING: Primary care clinics. PARTICIPANTS: A total of 22 primary care clinics across 7 provinces and 1 territory. MAIN OUTCOME MEASURES: Wait time 1, defined as the period between a patient's referral by a family physician to a specialist and the visit with said specialist. RESULTS: Overall, 2060 referrals initiated between January 2014 and December 2016 were included in the analysis. The median national wait time 1 was 78 days (interquartile range [IQR] of 34 to 175 days). The shortest waits were observed in Saskatchewan (51 days; IQR = 23 to 101 days) and British Columbia (59 days; IQR = 29 to 131 days), whereas the longest were in New Brunswick (105 days; IQR = 43 to 242 days) and Quebec (104 days; IQR = 36 to 239 days). Median wait time 1 varied substantially among different specialty groups, with the longest wait time for plastic surgery (159 days; IQR = 59 to 365 days) and the shortest for infectious diseases (14 days; IQR = 6 to 271 days). CONCLUSION: This is the first national examination of wait time 1 from the primary care perspective. It provides a picture of patient access to specialists across provinces and specialty groups. This research provides decision makers with important context for developing programs and policies aimed at addressing the largely ignored stage of the wait time continuum from the time of referral to eventual appointment time with the specialist.

Comprehensive analysis of all evolutionary paths between two divergent PDZ domain specificities.

To understand the molecular evolution of functional diversity in protein families, we comprehensively investigated the consequences of all possible mutation combinations separating two peptide-binding domains with highly divergent specificities. We analyzed the Erbin PDZ domain (Erbin-PDZ), which exhibits canonical type I specificity, and a synthetic Erbin-PDZ variant (E-14) that differs at six positions and exhibits an atypical specificity that closely resembles that of the natural Pdlim4 PDZ domain (Pdlim4-PDZ). We constructed a panel of 64 PDZ domains covering all possible transitions between Erbin-PDZ and E-14 (i.e., the panel contained variants with all possible combinations of either the Erbin-PDZ or E-14 sequence at the six differing positions). We assessed the specificity profiles of the 64 PDZ domains using a C-terminal phage-displayed peptide library containing all possible genetically encoded heptapeptides. The specificity profiles clustered into six distinct groups, showing that intermediate domains can be nodes for the evolution of divergent functions. Remarkably, three substitutions were sufficient to convert the specificity of Erbin-PDZ to that of Pdlim4-PDZ, whereas Pdlim4-PDZ contains 71 differences relative to Erbin-PDZ. X-ray crystallography revealed the structural basis for specificity transition: a single substitution in the center of the binding site, supported by contributions from auxiliary substitutions, altered the main chain conformation of the peptide ligand to resemble that of ligands bound to Pdlim4-PDZ. Our results show that a very small set of mutations can dramatically alter protein specificity, and these findings support the hypothesis whereby complex protein functions evolve by gene duplication followed by cumulative mutations.

Supporting the spread and scale-up of electronic consultation across Canada: cross-sectional analysis.

OBJECTIVE: To examine the process of implementing an electronic consultation (eConsult) service and evaluate its impact along key metrics outlined by the Reach, Effectiveness, Adoption, Implementation and Maintenance (RE-AIM) framework. DESIGN: Cross-sectional study. SETTING: Clinics using eConsult in four provinces across Canada: Alberta, Manitoba, Quebec and Newfoundland and Labrador. PARTICIPANTS: All eConsult cases submitted in four participating provinces were included. INTERVENTION: The eConsult service is a secure online application that allows primary care providers and specialists to communicate regarding a patient's care. We measured the impact using system utilisation data and mandatory close-out surveys completed at the end of each eConsult. MAIN OUTCOME MEASURES: Implementation progress and impact were examined using the five categories outlined by the RE-AIM framework: reach, effectiveness, adoption, implementation and maintenance. RESULTS: Four provinces provided data from different periods, ranging from 4 years (Alberta) to 10 months (Manitoba). Total cases completed ranged from 96 (Manitoba) to 6885 (Alberta). Newfoundland had the largest menu of available specialties (n=35), while Alberta and Quebec had the smallest (n=22). The most frequently requested groups varied across provinces, with only endocrinology appearing in the top five for all provinces. The average specialist response time ranged from 3 days (Manitoba) to 16.7 days (Alberta). Between 54% (Newfoundland) and 66% (Manitoba) of cases resulted in new or additional information. Primary care providers avoided completing referrals they had originally considered in 36% (Newfoundland) to 53% of cases (Manitoba), while only between 27 % (Quebec) and 29% (Newfoundland) of cases resulted in a referral. In every province, services demonstrated higher rates of usage in their last quarter of data than their first. CONCLUSIONS: eConsult was successfully implemented in four new provinces across Canada. Implementation strategies and scope varied, but services demonstrated substantial consistency on several key metrics, most notably on whether new information was learnt and impact on decision to refer.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.

Novel Aldo-Keto Reductases for the Biocatalytic Conversion of 3-Hydroxybutanal to 1,3-Butanediol: Structural and Biochemical Studies.

The nonnatural alcohol 1,3-butanediol (1,3-BDO) is a valuable building block for the synthesis of various polymers. One of the potential pathways for the biosynthesis of 1,3-BDO includes the biotransformation of acetaldehyde to 1,3-BDO via 3-hydroxybutanal (3-HB) using aldolases and aldo-keto reductases (AKRs). This pathway requires an AKR selective for 3-HB, but inactive toward acetaldehyde, so it can be used for one-pot synthesis. In this work, we screened more than 20 purified uncharacterized AKRs for 3-HB reduction and identified 10 enzymes with significant activity and nine proteins with detectable activity. PA1127 from Pseudomonas aeruginosa showed the highest activity and was selected for comparative studies with STM2406 from Salmonella enterica serovar Typhimurium, for which we have determined the crystal structure. Both AKRs used NADPH as a cofactor, reduced a broad range of aldehydes, and showed low activities toward acetaldehyde. The crystal structures of STM2406 in complex with cacodylate or NADPH revealed the active site with bound molecules of a substrate mimic or cofactor. Site-directed mutagenesis of STM2406 and PA1127 identified the key residues important for the activity against 3-HB and aromatic aldehydes, which include the residues of the substrate-binding pocket and C-terminal loop. Our results revealed that the replacement of the STM2406 Asn65 by Met enhanced the activity and the affinity of this protein toward 3-HB, resulting in a 7-fold increase in kcat/Km Our work provides further insights into the molecular mechanisms of the substrate selectivity of AKRs and for the rational design of these enzymes toward new substrates.IMPORTANCE In this study, we identified several aldo-keto reductases with significant activity in reducing 3-hydroxybutanal to 1,3-butanediol (1,3-BDO), an important commodity chemical. Biochemical and structural studies of these enzymes revealed the key catalytic and substrate-binding residues, including the two structural determinants necessary for high activity in the biosynthesis of 1,3-BDO. This work expands our understanding of the molecular mechanisms of the substrate selectivity of aldo-keto reductases and demonstrates the potential for protein engineering of these enzymes for applications in the biocatalytic production of 1,3-BDO and other valuable chemicals.

Assessing prescribing of NSAIDs, antiplatelets, and anticoagulants in Canadian family medicine using chart review.

Background Drug-related problems have been identified as a major contributor to emergency room visits, hospitalizations, and death. The most commonly implicated medications are nonsteroidal anti-inflammatory drugs (NSAIDs), antiplatelets, and anticoagulants. Considering a significant proportion of these harms are preventable, indicators to identify risky prescribing before they lead to harm have been developed. Objective To examine the prevalence and patterns of potentially inappropriate prescriptions (PIPs) in a primary care population who are using high-risk medications. Setting This study was performed within two multi-disciplinary family medicine teaching clinics in Winnipeg, Canada. Method A cross-sectional electronic/paper chart audit was conducted within two multi-disciplinary family medicine teaching clinics to evaluate the prevalence of 13 evidence-based high-risk prescriptions. Patients were included if they were prescribed an oral NSAID, antiplatelet, or an anticoagulant within the 12 month period between June 2012 and June 2013. Main outcome measure The proportion of PIPs associated with an increased bleeding risk for NSAIDs, antiplatelets, and anticoagulants. Results Of the 567 patients included in the review, 198 (35 %) patients had received at least 1 PIP in the past year. The most common PIP was the use of an oral NSAID with one or more GI risk factors without adequate gastro-protection. Only 34 (6 %) of these patients received a full medication review performed by a pharmacist. Although not statistically significant, patients who received a medication review had fewer inappropriate prescriptions (27 % with review, 35 % without). Conclusion Over one-third of the patients who were using high-risk medications were using them potentially inappropriately. Although pharmacists have been shown to reduce the amount of inappropriate prescribing, very few patients using these medications were referred to the pharmacist for a full medication review. These data suggest that there is opportunity for the identification and assessment of these patients when prescribing or dispensing these high-risk medications.

High risk use of OTC NSAIDs and ASA in family medicine: A retrospective chart review.

BACKGROUND: Complications associated with the use of NSAIDs, antiplatelet agents, and anticoagulants are among the top causes of preventable drug-related ER visits, hospitalizations and death. Although over-the-counter (OTC) NSAIDs and ASA also contribute to this preventable risk, it is unclear how well these medications are documented in primary care records. METHODS: A retrospective electronic and paper chart review was conducted to evaluate the prevalence of 13 evidence-based high-risk prescriptions and the contribution of OTC NSAIDs and ASA to these potentially inappropriate prescriptions (PIPs). RESULTS: Of the 148 patients included in the review, ASA was taken by 117 patients (79%) while OTC NSAIDs were taken by 36 (24%). OTC NSAIDs were never documented within the "medication" section of the electronic record, whereas ASA was documented in 65 (56%) cases. Eighty percent (118/148) taking either OTC NSAIDs or ASA were identified as having at least one PIP. CONCLUSION: OTC NSAIDs and ASA are widely available and are commonly taken without the knowledge of the prescriber. These medications contribute to the overall risk of bleeding. Review and documentation of OTC NSAIDs and ASA use should be part of all relevant patient encounters when prescribing NSAIDs, antiplatelets and anticoagulants.

Adopting electronic medical records: are they just electronic paper records?

OBJECTIVE: To understand the key challenges to adoption of advanced features of electronic medical records (EMRs) in office practice, and to better understand these challenges in a Canadian context. DESIGN: Mixed-methods study. SETTING: Manitoba. PARTICIPANTS: Health care providers and staff in 5 primary care offices. METHODS: Level of EMR adoption was assessed, and field notes from interviews and discussion groups were qualitatively analyzed for common challenges and themes across all sites. MAIN FINDINGS: Fifty-seven interviews and 4 discussion groups were conducted from November 2011 to January 2012. Electronic medical record adoption scores ranged from 2.3 to 3.0 (out of a theoretical maximum of 5). Practices often scored lower than expected on use of decision support, providing patients with access to their own data, and use of practice-reporting tools. Qualitative analysis showed there were ceiling effects to EMR adoption owing to how the EMR was implemented, the supporting eHealth infrastructure, lack of awareness or availability of EMR functionality, and poor EMR data quality. CONCLUSION: Many practitioners used their EMRs as "electronic paper records" and were not using advanced features of their EMRs that could further enhance practice. Data-quality issues within the EMRs could affect future attempts at using these features. Education and quality improvement activities to support data quality and EMR optimization are likely needed to support practices in maximizing their use of EMRs.

Structural analysis of HopPmaL reveals the presence of a second adaptor domain common to the HopAB family of Pseudomonas syringae type III effectors.

HopPmaL is a member of the HopAB family of type III effectors present in the phytopathogen Pseudomonas syringae. Using both X-ray crystallography and solution nuclear magnetic resonance, we demonstrate that HopPmaL contains two structurally homologous yet functionally distinct domains. The N-terminal domain corresponds to the previously described Pto-binding domain, while the previously uncharacterised C-terminal domain spans residues 308-385. While structurally similar, these domains do not share significant sequence similarity and most importantly demonstrate significant differences in key residues involved in host protein recognition, suggesting that each of them targets a different host protein.

Presentation and management of pediatric orbital cellulitis.

BACKGROUND: Orbital cellulitis is a serious, vision-threatening infection. OBJECTIVE: To review the epidemiology and clinical data of pediatric orbital cellulitis in Manitoba. METHODS: A 12-year retrospective review was conducted of all children (younger than 18 years of age) with orbital cellulitis admitted to Manitoba's only tertiary pediatric centre. Admission rates for orbital cellulitis were compared over three distinct time periods, based on licensure and funding levels of the heptavalent pneumococcal conjugate vaccine (PCV7) in Manitoba. RESULTS: Thirty-eight patients with orbital cellulitis were identified. Of these, 11% were of Aboriginal ethnicity in contrast with 30% to 40% of children who were admitted for other respiratory illnesses. Subperiosteal abscesses occurred in 31.5%. Only eight patients (21%) required surgery. Follow-up imaging after presentation usually did not indicate a need for subsequent surgical drainage. The mean number of orbital cellulitis cases per 1000 admissions for the following periods - before PCV7 licensure, after licensure and before full provincial funding, and after licensure and full funding - were 0.39, 0.53 and 0.90, respectively. No significant difference was noted among any of the periods as PCV7 coverage increased. CONCLUSIONS: The rate of subperiosteal abscesses was lower than other reports. This may be due to the median age at presentation. In contrast to admissions for most other respiratory infections at the Winnipeg Children's Hospital (Winnipeg, Manitoba), Aboriginal ethnicity was uncommon. Surprisingly, rates of admissions for orbital cellulitis appeared to show an increasing trend with increasing access to PCV7 in Manitoba, although overall the number of cases was very small. Studies into the changing microbiology of orbital cellulitis and sinusitis are warranted.

Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis.

Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.

Type III effector activation via nucleotide binding, phosphorylation, and host target interaction.

The Pseudomonas syringae type III effector protein avirulence protein B (AvrB) is delivered into plant cells, where it targets the Arabidopsis RIN4 protein (resistance to Pseudomonas maculicula protein 1 [RPM1]-interacting protein). RIN4 is a regulator of basal host defense responses. Targeting of RIN4 by AvrB is recognized by the host RPM1 nucleotide-binding leucine-rich repeat disease resistance protein, leading to accelerated defense responses, cessation of pathogen growth, and hypersensitive host cell death at the infection site. We determined the structure of AvrB complexed with an AvrB-binding fragment of RIN4 at 2.3 A resolution. We also determined the structure of AvrB in complex with adenosine diphosphate bound in a binding pocket adjacent to the RIN4 binding domain. AvrB residues important for RIN4 interaction are required for full RPM1 activation. AvrB residues that contact adenosine diphosphate are also required for initiation of RPM1 function. Nucleotide-binding residues of AvrB are also required for its phosphorylation by an unknown Arabidopsis protein(s). We conclude that AvrB is activated inside the host cell by nucleotide binding and subsequent phosphorylation and, independently, interacts with RIN4. Our data suggest that activated AvrB, bound to RIN4, is indirectly recognized by RPM1 to initiate plant immune system function.