Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase.

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(ß,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery.

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(ß,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

Rationally Designed Base Editors for Precise Editing of the Sickle Cell Disease Mutation.

Base editors are fusions of a deaminase and CRISPR-Cas ribonucleoprotein that allow programmable installment of transition mutations without double-strand DNA break intermediates. The breadth of potential base editing targets is frequently limited by the requirement of a suitably positioned Cas9 protospacer adjacent motif. To address this, we used structures of Cas9 and TadA to design a set of inlaid base editors (IBEs), in which deaminase domains are internal to Cas9. Several of these IBEs exhibit shifted editing windows and greater editing efficiency, enabling editing of targets outside the canonical editing window with reduced DNA and RNA off-target editing frequency. Finally, we show that IBEs enable conversion of the pathogenic sickle cell hemoglobin allele to the naturally occurring HbG-Makassar variant in patient-derived hematopoietic stem cells.

Directed evolution of adenine base editors with increased activity and therapeutic application.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity.

Cytosine base editors (CBEs) enable efficient, programmable reversion of T•A to C•G point mutations in the human genome. Recently, cytosine base editors with rAPOBEC1 were reported to induce unguided cytosine deamination in genomic DNA and cellular RNA. Here we report eight next-generation CBEs (BE4 with either RrA3F [wt, F130L], AmAPOBEC1, SsAPOBEC3B [wt, R54Q], or PpAPOBEC1 [wt, H122A, R33A]) that display comparable DNA on-target editing frequencies, whilst eliciting a 12- to 69-fold reduction in C-to-U edits in the transcriptome, and up to a 45-fold overall reduction in unguided off-target DNA deamination relative to BE4 containing rAPOBEC1. Further, no enrichment of genome-wide C•G to T•A edits are observed in mammalian cells following transfection of mRNA encoding five of these next-generation editors. Taken together, these next-generation CBEs represent a collection of base editing tools for applications in which minimized off-target and high on-target activity are required.

Author Correction: Phenotypes associated with genes encoding drug targets are predictive of clinical trial side effects.

In the original version of this article, there were errors in the labelling of the colours in the key of Figure 2, whereby the labeling of the third and fourth of the four colours was reversed. This has been corrected in both the PDF and HTML versions of the article.

Phenotypes associated with genes encoding drug targets are predictive of clinical trial side effects.

Only a small fraction of early drug programs progress to the market, due to safety and efficacy failures, despite extensive efforts to predict safety. Characterizing the effect of natural variation in the genes encoding drug targets should present a powerful approach to predict side effects arising from drugging particular proteins. In this retrospective analysis, we report a correlation between the organ systems affected by genetic variation in drug targets and the organ systems in which side effects are observed. Across 1819 drugs and 21 phenotype categories analyzed, drug side effects are more likely to occur in organ systems where there is genetic evidence of a link between the drug target and a phenotype involving that organ system, compared to when there is no such genetic evidence (30.0 vs 19.2%; OR = 1.80). This result suggests that human genetic data should be used to predict safety issues associated with drug targets.

Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase.

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We herein provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, of which several members are critical for the virulence of pathogenic mycobacteria.

Structural basis for methylphosphonate biosynthesis.

Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from Streptomyces albus (SaHEPD), and find that it displays the same motif. SaHEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant Pelagibacter ubique. The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus.

Sikhs in Minnesota: Dental Care Access, Ethnicity, and Acculturation.

To explore the dental care experiences of one of Minnesota's ethnic communities, the authors conducted pilot study among members of Sikh temple. The Sikhs, a religious group from the Punjab region of India, number roughly 1,500-2,000 individuals in the Twin Cities. Forty-seven adults from the Bloomington, Minnesot temple completed the survey, which was administered during interviews with one of the authors, himself a Sikh. Respondents, who ranged in age from 29 to 92, have been in the United States for an average of 16 years, and had educational backgrounds ranging from undergraduate, Masters, to doctoral degrees. Their self-reported oral health is deemed good. They regularly seek dental care, including preventive services. Their experiences with dental practices have largely been positive, and they have found little discrimination when encountering dental office staff members. Some degree of communication issues was reported by 25% of respondents. Roughly half of the persons surveyed seek care in private practices, but a surprisingly high percentage (13%) visit the University of Minnesota School of Dentistry clinics. Most respondents were comfortable discussing their culture and religion with dental office staff members, and they seemed to hold a positive view of Minnesota's dental profession. Sikh weddings are always full of rituals, traditions, and celebrations. The wedding usually takes place in the morning and is held in front of the Sikh holy book Shri Guru Granth Sahib. A bride may traditionally be dressed in red, as the color red symbolizes love and commitment in Indian culture. The groom wears a colored turban and scarf and carries a kirpan, a ceremonial sword a dagger. The priest leading this ceremony explains the Sikh philosophy of marriage. Family members, relatives, and friends gather to congratulate and bless the new couple, and the whole wedding can last three to four days, made up of a number of rituals.

Structural Basis for Substrate Specificity in Adenosylcobalamin-dependent Isobutyryl-CoA Mutase and Related Acyl-CoA Mutases.

Acyl-CoA mutases are a growing class of adenosylcobalamin-dependent radical enzymes that perform challenging carbon skeleton rearrangements in primary and secondary metabolism. Members of this class of enzymes must precisely control substrate positioning to prevent oxidative interception of radical intermediates during catalysis. Our understanding of substrate specificity and catalysis in acyl-CoA mutases, however, is incomplete. Here, we present crystal structures of IcmF, a natural fusion protein variant of isobutyryl-CoA mutase, in complex with the adenosylcobalamin cofactor and four different acyl-CoA substrates. These structures demonstrate how the active site is designed to accommodate the aliphatic acyl chains of each substrate. The structures suggest that a conformational change of the 5'-deoxyadenosyl group from C2'-endo to C3'-endo could contribute to initiation of catalysis. Furthermore, detailed bioinformatic analyses guided by our structural findings identify critical determinants of acyl-CoA mutase substrate specificity and predict new acyl-CoA mutase-catalyzed reactions. These results expand our understanding of the substrate specificity and the catalytic scope of acyl-CoA mutases and could benefit engineering efforts for biotechnological applications ranging from production of biofuels and commercial products to hydrocarbon remediation.

Dental therapy: evolving in Minnesota's safety net.

OBJECTIVES: We identified Minnesota's initial dental therapy employers and surveyed dental safety net providers' perceptions of dental therapy. METHODS: In July 2011, we surveyed 32 Minnesota dental safety net providers to assess their prospective views on dental therapy employment options. In October 2013, we used an employment scan to reveal characteristics of the early adopters of dental therapy. RESULTS: Before the availability of licensed dental therapists, safety net dental clinic directors overwhelmingly (77%) supported dental therapy. As dental therapists have become licensed over the past 2 years, the early employers of dental therapists are safety net clinics. CONCLUSIONS: Although the concept of dental therapy remains controversial in Minnesota, it now has a firm foundation in the state's safety net clinics. Dental therapists are being used in innovative and diverse ways, so, as dental therapy continues to evolve, further research to identify best practices for incorporating dental therapists into the oral health care team is needed.

Beginning the socialization to a new workforce model: dental students' preliminary knowledge of and attitudes about the role of the dental therapist.

The purpose of the study reported here was to assess first- and second-year dental students' knowledge of and attitudes about the role of the dental therapist in the oral health care delivery system. The results of this study are informing the continued development and implementation of a new dental workforce training model at the University of Minnesota. Dental students at the university (Classes of 2012 and 2013) were surveyed in 2009, with follow-up surveys planned for the subsequent five years. Multiple-choice questions and statements to be ranked using a Likert scale were used to determine what the students knew and thought about dental therapists' scope of practice, care delivery, work quality, cost-effectiveness, and role in reducing disparities in oral health care access. The results suggest that the students had generally neutral or uncertain attitudes about dental therapy, based on minimal knowledge about the role of dental therapists. In addition, we found little difference in attitudes between the two classes, the only exception being that the first-year students less often perceived the therapists as a solution to access problems. These baseline data are guiding the intraprofessional training of dental, dental hygiene, and dental therapy students toward the goal of positive socialization to a new workforce model and affirmation of the dental therapist as a member of the oral health care team.

Dental hygiene workforce issues: a Minnesota study.

PURPOSE: This study was designed to explore the prevalence and reasons for withdrawal from the dental hygiene workforce. The study also assessed the reasons for maintaining an active license after withdrawal, and the factors that might entice an individual to return to the dental hygiene workforce. METHODS: A random sample was selected of dental hygienists licensed in Minnesota from the November 2003 State Board of Dentistry's dental hygiene licensing file. Data was collected from a mailed questionnaire containing 45 open-ended and close-ended questions. The final sample included 2672 dental hygienists actively licensed and residing in Minnesota. One thousand four hundred and twenty responded to the survey, for a response rate of 53%. From that sample, 129 participants indicated that they were licensed to practice in Minnesota but were not currently working (had withdrawn from the workforce). This study focuses on the responses of those 129 individuals. RESULTS: The prevalence of withdrawal was 9%, with the majority of the participants indicating that they did not plan to return to the dental hygiene workforce. The primary reason for withdrawal was child-rearing responsibilities, followed by health-related reasons, and pursuing a different career. A key difference between those who were active participants in the workforce and those who had withdrawn was related to income. The results of this study found that financial difficulties, death/disability of a spouse, and having older children were the primary factors that would entice a respondent to return as an active participant to the dental hygiene workforce. CONCLUSION: This study suggests that dental hygiene workforce issues are of great complexity and are multifactorial in nature. The findings tend to confirm the results of previous work. It can be concluded that because of the nature of the work and the demands of the profession, it is likely that movement in and out of the dental hygiene workforce will continue to be an issue in the profession.

Self-reported toothache experience in an adult population in Benin City, Edo State, Nigeria.

PURPOSE: This pilot study was conducted to estimate the prevalence of self-reported toothache, bleeding gums and oral ulcer experiences, reasons for dental visits and associated sociodemographic factors among adults living in Benin City, Edo State, Nigeria. MATERIALS AND METHODS: This cross-sectional study was based on a convenience sample in an urban setting. Out of the 540 persons surveyed, 508 had complete usable information, 48% males and 52% females, and aged 18 - 54 years. A closed questionnaire was used to collect the data at two large medical outpatient facilities and a university community. RESULTS: The proportion reporting toothache experience in the previous 12 months was 34%; painful/ bleeding gums 28%, and oral ulcers/painful spots 14%. Univariate analysis showed that toothache experience was associated with time since last dental visit (p < 0.001), age (p < 0.001), gender (p < 0.001) and education (p < 0.001). Experience with bleeding gums was associated with gender (p < 0.001), education (p < 0.001) and last dental visits (p < 0.001). Oral ulcers were associated with gender (p = 0.004), age (p < 0.001) and last dental visits (p < 0.001). Fewer males than females reported toothache (25% vs. 42%, OR 1.65) but more males reported bleeding gums (37% vs. 19%, OR = 0.34) and oral ulcers (19% vs. 10% OR = 0.33). CONCLUSIONS: Toothache experience was the most prevalent oral health problem reported by adults. Oral health problems were associated with age, gender and last dental visits.

Self-reported dental visits among adults in Benin City, Nigeria.

OBJECTIVE: To identify patterns and predictive factors independently associated with self-reported dental visits of adults resident in Benin City, Edo State, Nigeria. METHODS: A cross-sectional study of 358 adults aged 18-64 years using a closed ended, oral health questionnaire administered by trained interviewers to assess sociodemographic information and factors associated with oral healthcare utilisation. RESULTS: 26% of adults reported having visited a dentist within the previous 12 months, 22% within 12-36 months, 30% over 36 months previously, and 22% had never been to a dentist. 32% visited a dentist for treatment, 22% when in pain, and 2% when referred. Those aged 25-34 years were twice as likely as those aged 18-24 years to have visited a dentist in the past year after adjusting for sex, and employment. Women were twice as likely as men to have visited a dentist within the past 12 months after adjusting for age and employment. CONCLUSION: Being of younger age, female and employed were associated with visiting a dentist in the previous 12 months.