Autoinhibition and Signaling by the Switch II Motif in the G-protein Chaperone of a Radical B12 Enzyme

MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5′-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlF_x⁻ to form the putative transition state analog, GDP·AlF₄⁻. The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.     

Half of Pulmonary Tuberculosis Cases Were Left Undiagnosed in Prisons of the Tigray Region of Ethiopia: Implications for Tuberculosis Control

Introduction

Prison settings have been often identified as important but neglected reservoirs for TB. This study was designed to determine the prevalence of undiagnosed pulmonary TB and assess the potential risk factors for such TB cases in prisons of the Tigray region.

Method

A cross-sectional study was conducted between August 2013 and February 2014 in nine prisons. A standardized symptom-based questionnaire was initially used to identify presumptive TB cases. From each, three consecutive sputum samples were collected for acid-fast bacilli (AFB) microscopy and culture. Blood samples were collected from consented participants for HIV testing.

Result

Out of 809 presumptive TB cases with culture result, 4.0% (95% CI: 2.65–5.35) were confirmed to have undiagnosed TB. The overall estimated point prevalence of undiagnosed TB was found to be 505/100,000 prisoners (95% CI: 360–640). Together with the 27 patients who were already on treatment, the overall estimated point prevalence of TB would be 793/100,000 prisoners (95% CI: 610–970), about four times higher than in the general population. The ratio of active to passive case detection was 1.18:1. The prevalence of HIV was 4.4% (36/809) among presumptive TB cases and 6.3% (2/32) among undiagnosed TB cases. In a multivariate logistic regression analysis, chewing Khat (adjusted OR = 2.81; 95% CI: 1.02–7.75) and having had a close contact with a TB patient (adjusted OR = 2.18; 95% CI: 1.05–4.51) were found to be predictors of undiagnosed TB among presumptive TB cases.

Conclusions

This study revealed that at least half of symptomatic pulmonary TB cases in Northern Ethiopian prisons remain undiagnosed and hence untreated. The prevalence of undiagnosed TB in the study prisons was more than two folds higher than in the general population of Tigray. This may indicate the need for more investment and commitment to improving TB case detection in the study prisons.     

Lactate threshold and performance adaptations to 4 weeks of training in untrained swimmers: volume vs. intensity

The purpose of this study was to investigate the impact of 4 weeks of high-intensity vs. high-volume swim training on lactate threshold (LT) characteristics and performance. Thirteen untrained swimmers with a mean age of 19.0 ± 0.5 undertook an incremental swimming test before and after 4 weeks of training for the determination of LT. Performance was evaluated by a 50-m maximum freestyle test. The swimmers were assigned to 1 of each of 2 training groups. The high-intensity group (n = 6) focused on sprint training (SP) and swam a total of 1,808 ± 210 m. The high-volume group (n = 7) followed the same program as the SP group but swam an additional 1,100 m (38% more) of endurance swimming (SP + End). A training effect was evident in both groups as seen by the similar improvements in sprint performance of the 50-m maximum time (p < 0.01), peak velocity increases and the lower value of lactate at the individual LTs (p < 0.01). Lactate threshold velocity improved only in the SP + End group from 1.20 ± 0.12 m·s(-1) pretraining to 1.32 ± 0.12 m·s(-1) posttraining (p = 0.77, effect size = 1, p < 0.01), expressed by the rightward shifts of the individual lactate-velocity curves, indicating an improvement in the aerobic capacity. Peak lactate and lactate concentrations at LT did not significantly change. In conclusion, this study was able to demonstrate that 4 weeks of either high-intensity or high-volume training was able to demonstrate similar improvements in swimming performance. In the case of lack of significant changes in lactate profiling in response to high-intensity training, we could suggest a dissociation between the 2.     

Structural basis of multifunctionality in a vitamin B12-processing enzyme

An early step in the intracellular processing of vitamin B(12) involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B(12)), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPH-dependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.     

Molecular basis of actin nucleation factor cooperativity: crystal structure of the Spir-1 kinase non-catalytic C-lobe domain (KIND)•formin-2 formin SPIR interaction motif (FSI) complex

The distinct actin nucleation factors of the Spir and formin subgroup families cooperate in actin nucleation. The Spir/formin cooperativity has been identified to direct two essential steps in mammalian oocyte maturation, the asymmetric spindle positioning and polar body extrusion during meiosis. Understanding the nature and regulation of the Spir/Fmn cooperation is an important requirement to comprehend mammalian reproduction. Recently we dissected the structural elements of the Spir and Fmn family proteins, which physically link the two actin nucleation factors. The trans-regulatory interaction is mediated by the Spir kinase non-catalytic C-lobe domain (KIND) and the C-terminal formin Spir interaction motif (FSI). The interaction inhibits formin nucleation activity and enhances the Spir activity. To get insights into the molecular mechanism of the Spir/Fmn interaction, we determined the crystal structure of the KIND domain alone and in complex with the C-terminal Fmn-2 FSI peptide. Together they confirm the proposed structural homology of the KIND domain to the protein kinase fold and reveal the basis of the Spir/formin interaction. The complex structure showed a large interface with conserved and positively charged residues of the Fmn FSI peptide mediating major contacts to an acidic groove on the surface of KIND. Protein interaction studies verified the electrostatic nature of the interaction. The data presented here provide the molecular basis of the Spir/formin interaction and give a first structural view into the mechanisms of actin nucleation factor cooperativity.     

Specific potassium ion interactions facilitate homocysteine binding to betaine‐homocysteine S‐methyltransferase

Betaine‐homocysteine S‐methyltransferase (BHMT) is a zinc‐dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S‐adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K_M for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K_M of the enzyme for homocysteine, but it does not affect the apparent K_M for betaine or the apparent k_cat for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26‐Gly27‐Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site‐specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme. Proteins 2014; 82:2552–2564. © 2014 Wiley Periodicals, Inc.     

Evaluation of an intact,an ACL-deficient,and a reconstructed human knee joint finite element model

The human knee joint has a three-dimensional geometry with multiple body articulations that produce complex mechanical responses under loads that occur in everyday life and sports activities. Understanding the complex mechanical interactions of these load-bearing structures is of use when the treatment of relevant diseases is evaluated and assisting devices are designed. The anterior cruciate ligament (ACL) in the knee is one of four main ligaments that connects the femur to the tibia and is often torn during sudden twisting motions, resulting in knee instability. The objective of this work is to study the mechanical behavior of the human knee joint and evaluate the differences in its response for three different states, i.e., intact, ACL-deficient, and surgically treated (reconstructed) knee. The finite element models corresponding to these states were developed. For the reconstructed model, a novel repair device was developed and patented by the author in previous work. Static load cases were applied, as have already been presented in a previous work, in order to compare the calculated results produced by the two models the ACL-deficient and the surgically reconstructed knee joint, under the exact same loading conditions. Displacements were calculated in different directions for the load cases studied and were found to be very close to those from previous modeling work and were in good agreement with experimental data presented in literature. The developed finite element model for both the intact and the ACL-deficient human knee joint is a reliable tool to study the kinematics of the human knee, as results of this study show. In addition, the reconstructed human knee joint model had kinematic behavior similar to the intact knee joint, showing that such reconstruction devices can restore human knee stability to an adequate extent.     

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping,Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.