Shaping Perceptions and Inspiring Future Neurosurgeons: The Value of a Hands-On Simulated Aneurysm Clipping Workshops at a Student-Organized Neurosurgical Conference.

Objective Early exposure to niche specialities, like neurosurgery, is essential to inform decisions about future training in these specialities. This study assesses the impact of a hands-on simulated aneurysm clipping workshop on medical students' and junior doctors' perceptions of neurosurgery at a student-organized neurosurgical conference. Methods Ninety-six delegates were sampled from a hands-on workshop involving hydrogel three-dimensional printed aneurysms clipping using surgical microscopes. Consultant neurosurgeons facilitated the workshop. Changes in delegates' perceptions of neurosurgery were collected using Likert scale and free-text responses postconference. Results Postworkshop, 82% of participants reported a positive impact on their perception of neurosurgery. Thematic analysis revealed that delegates valued the hands-on experience, exposure to microsurgery, and interactions with consultant neurosurgeons. Thirty-six of the 96 delegates (37.5%) expressed that the workshop dispelled preconceived fears surrounding neurosurgery and improved understanding of a neurosurgeon's day-to-day tasks. Several delegates initially apprehensive about neurosurgery were now considering it as a career. Conclusion Hands-on simulated workshops can effectively influence medical students' and junior doctors' perceptions of neurosurgery, providing valuable exposure to the specialty. By providing a valuable and immersive introduction to the specialty, these workshops can help to dispel misconceptions, fears, and apprehensions associated with neurosurgery, allowing them to consider the specialty to a greater degree than before. This study of a one-time workshop cannot effectively establish its long-term impact on said perceptions, however.

A treatable inborn error of metabolism presenting in the sixth decade.

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. If untreated, PKU can result in global developmental delay, learning difficulties or seizures. For that reason, PKU is included in the UK neonatal screening programme. We describe a patient in his sixth decade presenting with progressive cognitive decline and spasticity, in whom a diagnosis of PKU was eventually reached. We note that although we currently have a robust neonatal screening programme, this has not always been the case. Patients born before 1969 were not screened, and tests used in early screening programmes were less sensitive than those used today. This case serves as a reminder that inherited metabolic disorders may present in later life and may mimic the neurocognitive and radiological picture of other white matter syndromes.

CSF Findings in Relation to Clinical Characteristics, Subtype, and Disease Course in Patients With Guillain-Barré Syndrome.

BACKGROUND AND OBJECTIVES: To investigate CSF findings in relation to clinical and electrodiagnostic subtypes, severity, and outcome of Guillain-Barré syndrome (GBS) based on 1,500 patients in the International GBS Outcome Study. METHODS: Albuminocytologic dissociation (ACD) was defined as an increased protein level (>0.45 g/L) in the absence of elevated white cell count (<50 cells/µL). We excluded 124 (8%) patients because of other diagnoses, protocol violation, or insufficient data. The CSF was examined in 1,231 patients (89%). RESULTS: In 846 (70%) patients, CSF examination showed ACD, which increased with time from weakness onset: ≤4 days 57%, >4 days 84%. High CSF protein levels were associated with a demyelinating subtype, proximal or global muscle weakness, and a reduced likelihood of being able to run at week 2 (odds ratio [OR] 0.42, 95% CI 0.25-0.70; p = 0.001) and week 4 (OR 0.44, 95% CI 0.27-0.72; p = 0.001). Patients with the Miller Fisher syndrome, distal predominant weakness, and normal or equivocal nerve conduction studies were more likely to have lower CSF protein levels. CSF cell count was <5 cells/µL in 1,005 patients (83%), 5-49 cells/µL in 200 patients (16%), and ≥50 cells/µL in 13 patients (1%). DISCUSSION: ACD is a common finding in GBS, but normal protein levels do not exclude this diagnosis. High CSF protein level is associated with an early severe disease course and a demyelinating subtype. Elevated CSF cell count, rarely ≥50 cells/µL, is compatible with GBS after a thorough exclusion of alternative diagnoses. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that CSF ACD (defined by the Brighton Collaboration) is common in patients with GBS.

An International Perspective on Preceding Infections in Guillain-Barré Syndrome: The IGOS-1000 Cohort.

BACKGROUND AND OBJECTIVES: Infections play a key role in the development of Guillain-Barré syndrome (GBS) and have been associated with specific clinical features and disease severity. The clinical variation of GBS across geographical regions has been suggested to be related to differences in the distribution of preceding infections, but this has not been studied on a large scale. METHODS: We analyzed the first 1,000 patients included in the International GBS Outcome Study with available biosamples (n = 768) for the presence of a recent infection with Campylobacter jejuni, hepatitis E virus, Mycoplasma pneumoniae, cytomegalovirus, and Epstein-Barr virus. RESULTS: Serologic evidence of a recent infection with C. jejuni was found in 228 (30%), M. pneumoniae in 77 (10%), hepatitis E virus in 23 (3%), cytomegalovirus in 30 (4%), and Epstein-Barr virus in 7 (1%) patients. Evidence of more than 1 recent infection was found in 49 (6%) of these patients. Symptoms of antecedent infections were reported in 556 patients (72%), and this proportion did not significantly differ between those testing positive or negative for a recent infection. The proportions of infections were similar across continents. The sensorimotor variant and the demyelinating electrophysiologic subtype were most frequent across all infection groups, although proportions were significantly higher in patients with a cytomegalovirus and significantly lower in those with a C. jejuni infection. C. jejuni-positive patients were more severely affected, indicated by a lower Medical Research Council sum score at nadir (p = 0.004) and a longer time to regain the ability to walk independently (p = 0.005). The pure motor variant and axonal electrophysiologic subtype were more frequent in Asian compared with American or European C. jejuni-positive patients (p < 0.001, resp. p = 0.001). Time to nadir was longer in the cytomegalovirus-positive patients (p = 0.004). DISCUSSION: Across geographical regions, the distribution of infections was similar, but the association between infection and clinical phenotype differed. A mismatch between symptom reporting and serologic results and the high frequency of coinfections demonstrate the importance of broad serologic testing in identifying the most likely infectious trigger. The association between infections and outcome indicates their value for future prognostic models.

Predicting Outcome in Guillain-Barré Syndrome: International Validation of the Modified Erasmus GBS Outcome Score.

BACKGROUND AND OBJECTIVES: The clinical course and outcome of the Guillain-Barré syndrome (GBS) are diverse and vary among regions. The modified Erasmus GBS Outcome Score (mEGOS), developed with data from Dutch patients, is a clinical model that predicts the risk of walking inability in patients with GBS. The study objective was to validate the mEGOS in the International GBS Outcome Study (IGOS) cohort and to improve its performance and region specificity. METHODS: We used prospective data from the first 1,500 patients included in IGOS, aged ≥6 years and unable to walk independently. We evaluated whether the mEGOS at entry and week 1 could predict the inability to walk unaided at 4 and 26 weeks in the full cohort and in regional subgroups, using 2 measures for model performance: (1) discrimination: area under the receiver operating characteristic curve (AUC) and (2) calibration: observed vs predicted probability of being unable to walk independently. To improve the model predictions, we recalibrated the model containing the overall mEGOS score, without changing the individual predictive factors. Finally, we assessed the predictive ability of the individual factors. RESULTS: For validation of mEGOS at entry, 809 patients were eligible (Europe/North America [n = 677], Asia [n = 76], other [n = 56]), and 671 for validation of mEGOS at week 1 (Europe/North America [n = 563], Asia [n = 65], other [n = 43]). AUC values were >0.7 in all regional subgroups. In the Europe/North America subgroup, observed outcomes were worse than predicted; in Asia, observed outcomes were better than predicted. Recalibration improved model accuracy and enabled the development of a region-specific version for Europe/North America (mEGOS-Eu/NA). Similar to the original mEGOS, severe limb weakness and higher age were the predominant predictors of poor outcome in the IGOS cohort. DISCUSSION: mEGOS is a validated tool to predict the inability to walk unaided at 4 and 26 weeks in patients with GBS, also in countries outside the Netherlands. We developed a region-specific version of mEGOS for patients from Europe/North America. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that the mEGOS accurately predicts the inability to walk unaided at 4 and 26 weeks in patients with GBS. TRIAL REGISTRATION INFORMATION: NCT01582763.

Current treatment practice of Guillain-Barré syndrome.

OBJECTIVE: To define the current treatment practice of Guillain-Barré syndrome (GBS). METHODS: The study was based on prospective observational data from the first 1,300 patients included in the International GBS Outcome Study. We described the treatment practice of GBS in general, and for (1) severe forms (unable to walk independently), (2) no recovery after initial treatment, (3) treatment-related fluctuations, (4) mild forms (able to walk independently), and (5) variant forms including Miller Fisher syndrome, taking patient characteristics and hospital type into account. RESULTS: We excluded 88 (7%) patients because of missing data, protocol violation, or alternative diagnosis. Patients from Bangladesh (n = 189, 15%) were described separately because 83% were not treated. IV immunoglobulin (IVIg), plasma exchange (PE), or other immunotherapy was provided in 941 (92%) of the remaining 1,023 patients, including patients with severe GBS (724/743, 97%), mild GBS (126/168, 75%), Miller Fisher syndrome (53/70, 76%), and other variants (33/40, 83%). Of 235 (32%) patients who did not improve after their initial treatment, 82 (35%) received a second immune modulatory treatment. A treatment-related fluctuation was observed in 53 (5%) of 1,023 patients, of whom 36 (68%) were re-treated with IVIg or PE. CONCLUSIONS: In current practice, patients with mild and variant forms of GBS, or with treatment-related fluctuations and treatment failures, are frequently treated, even in absence of trial data to support this choice. The variability in treatment practice can be explained in part by the lack of evidence and guidelines for effective treatment in these situations.

An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

Regional variation of Guillain-Barré syndrome.

Guillain-Barré syndrome is a heterogeneous disorder regarding the clinical presentation, electrophysiological subtype and outcome. Previous single country reports indicate that Guillain-Barré syndrome may differ among regions, but no systematic comparative studies have been conducted. Comparative studies are required to identify factors determining disease susceptibility, variation and prognosis, and to improve diagnostic criteria. The International Guillain-Barré Syndrome Outcome Study is a prospective, observational cohort study including all patients within the diagnostic spectrum, aiming to describe the heterogeneity of Guillain-Barré syndrome worldwide. The current study was based on the first 1000 inclusions with a follow-up of at least 1 year and confirmed the variation in clinical presentation, course and outcome between patients. The full clinical spectrum of Guillain-Barré syndrome was observed in patients from all countries participating in the International Guillain-Barré Syndrome Outcome Study, but the frequency of variants differed between regions. We compared three regions based on geography, income and previous reports of Guillain-Barré syndrome subtypes: 'Europe/Americas', 'Asia' (without Bangladesh), and 'Bangladesh'. We excluded 75 (8%) patients because of alternative diagnoses, protocol violations, or missing data. The predominant clinical variant was sensorimotor in Europe/Americas (n = 387/562, 69%) and Asia (n = 27/63, 43%), and pure motor in Bangladesh (n = 74/107, 69%). Miller Fisher syndrome and Miller Fisher-Guillain-Barré overlap syndrome were more common in Asia (n = 14/63, 22%) than in the other two regions (Europe/Americas: n = 64/562, 11%; Bangladesh: n = 1/107, 1%) (P < 0.001). The predominant electrophysiological subtype was demyelinating in all regions (Europe/Americas: n = 312/573, 55%; Asia: n = 29/65, 45%; Bangladesh: n = 38/94, 40%). The axonal subtype occurred more often in Bangladesh (n = 34/94, 36%) than in Europe/Americas (n = 33/573, 6%) and other Asian countries (n = 4/65, 6%) (P < 0.001). In all regions, patients with the axonal subtype were younger, had fewer sensory deficits, and showed a trend towards poorer recovery compared to patients with the demyelinating subtype. The proportion of patients able to walk unaided after 1 year varied between Asia (n = 31/34, 91%), Europe/Americas (n = 334/404, 83%) and Bangladesh (n = 67/97, 69%) (P = 0.003). A similar variation was seen for mortality, being higher in Bangladesh (n = 19/114, 17%) than in Europe/Americas (n = 23/486, 5%) and Asia (n = 1/45, 2%) (P < 0.001). This study showed that factors related to geography have a major influence on clinical phenotype, disease severity, electrophysiological subtype, and outcome of Guillain-Barré syndrome.

Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 µM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

Brainstem encephalitis and acute polyneuropathy associated with hepatitis E infection.

A 59-year-old man presented with feverish illness. His Glasgow Coma Scale was 15, had reduced visual acuity in the left eye with partial left ptosis and mild left hemiparesis with an extensor left plantar. Over 48 hours, he accrued multiple cranial nerves palsies and progressed to a flaccid paralysis necessitating admission to an intensive care unit.Cerebrospinal fluid (CSF) study showed 20 lymphocytes and raised protein. Viral and bacterial PCRs were negative. Samples for Lyme, blood-borne viruses, syphilis and autoantibodies were also negative. MRI brain showed T2 abnormalities within the brainstem. Nerve conduction studies revealed an acute motor and sensory axonal neuropathy pattern of Guillian Barre Syndrome (GBS). The patient was treated for both infective and inflammatory causes of brainstem encephalitis and GBS.Retrospective studies confirmed the presence of hepatitis E virus (HEV) RNA in CSF and serum studies showed positive HEV IgG and IgM prior to intravenous infusion. After 3 months of intensive rehabilitation, the patient was discharged home walking with a frame.

Inhibition of complement in Guillain-Barré syndrome: the ICA-GBS study.

The outcome of Guillain-Barré syndrome (GBS) remains unchanged since plasma exchange and intravenous immunoglobulin (IVIg) were introduced over 20 years ago. Pathogenesis studies on GBS have identified the terminal component of complement cascade as a key disease mediator and therapeutic target. We report the first use of terminal complement pathway inhibition with eculizumab in humans with GBS. In a randomised, double-blind, placebo-controlled trial, 28 subjects eligible on the basis of GBS disability grade of at least 3 were screened, of whom 8 (29%) were randomised. Five received eculizumab for 4 weeks, alongside standard IVIg treatment. The safety outcomes, monitored via adverse events capture, showed eculizumab to be well-tolerated and safe when administered in conjunction with IVIg. Primary and secondary efficacy outcomes in the form of GBS disability scores (GBS DS), MRC sum scores, Rasch overall disability scores, and overall neuropathy limitation scores are reported descriptively. For the primary efficacy outcome at 4 weeks after recruitment, two of two placebo- and two of five eculizumab-treated subjects had improved by one or more grades on the GBS DS. Although the small sample size precludes a statistically meaningful analysis, these pilot data indicate further studies on complement inhibition in GBS are warranted.

Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae.

The FtsEX:PcsB complex forms a molecular machine that carries out peptidoglycan (PG) hydrolysis during normal cell division of the major respiratory pathogenic bacterium, Streptococcus pneumoniae (pneumococcus). FtsX is an integral membrane protein and FtsE is a cytoplasmic ATPase that together structurally resemble ABC transporters. Instead of transport, FtsEX transduces signals from the cell division apparatus to stimulate PG hydrolysis by PcsB, which interacts with extracellular domains of FtsX. Structural studies of PcsB and one extracellular domain of FtsX have recently appeared, but little is known about the biochemical properties of the FtsE ATPase or the intact FtsX transducer protein. We report here purifications and characterizations of tagged FtsX and FtsE proteins. Pneumococcal FtsX-GFP-His and FtsX-His could be overexpressed in Escherichia coli without toxicity, and FtsE-His remained soluble during purification. FtsX-His dimerizes in detergent micelles and when reconstituted in phospholipid nanodiscs. FtsE-His binds an ATP analog with an affinity comparable to that of ATPase subunits of ABC transporters, and FtsE-His preparations have a low, detectable ATPase activity. However, attempts to detect complexes of purified FtsX-His, FtsE-His, and PcsB-His or coexpressed tagged FtsX and FtsE were not successful with the constructs and conditions tested so far. In working with nanodiscs, we found that PcsB-His has an affinity for charged phospholipids, mediated partly by interactions with its coiled-coil domain. Together, these findings represent first steps toward reconstituting the FtsEX:PcsB complex biochemically and provide information that may be relevant to the assembly of the complex on the surface of pneumococcal cells.

Vertebral Pneumaticity in the Ornithomimosaur Archaeornithomimus (Dinosauria: Theropoda) Revealed by Computed Tomography Imaging and Reappraisal of Axial Pneumaticity in Ornithomimosauria.

Among extant vertebrates, pneumatization of postcranial bones is unique to birds, with few known exceptions in other groups. Through reduction in bone mass, this feature is thought to benefit flight capacity in modern birds, but its prevalence in non-avian dinosaurs of variable sizes has generated competing hypotheses on the initial adaptive significance of postcranial pneumaticity. To better understand the evolutionary history of postcranial pneumaticity, studies have surveyed its distribution among non-avian dinosaurs. Nevertheless, the degree of pneumaticity in the basal coelurosaurian group Ornithomimosauria remains poorly known, despite their potential to greatly enhance our understanding of the early evolution of pneumatic bones along the lineage leading to birds. Historically, the identification of postcranial pneumaticity in non-avian dinosaurs has been based on examination of external morphology, and few studies thus far have focused on the internal architecture of pneumatic structures inside the bones. Here, we describe the vertebral pneumaticity of the ornithomimosaur Archaeornithomimus with the aid of X-ray computed tomography (CT) imaging. Complementary examination of external and internal osteology reveals (1) highly pneumatized cervical vertebrae with an elaborate configuration of interconnected chambers within the neural arch and the centrum; (2) anterior dorsal vertebrae with pneumatic chambers inside the neural arch; (3) apneumatic sacral vertebrae; and (4) a subset of proximal caudal vertebrae with limited pneumatic invasion into the neural arch. Comparisons with other theropod dinosaurs suggest that ornithomimosaurs primitively exhibited a plesiomorphic theropod condition for axial pneumaticity that was extended among later taxa, such as Archaeornithomimus and large bodied Deinocheirus. This finding corroborates the notion that evolutionary increases in vertebral pneumaticity occurred in parallel among independent lineages of bird-line archosaurs. Beyond providing a comprehensive view of vertebral pneumaticity in a non-avian coelurosaur, this study demonstrates the utility and need of CT imaging for further clarifying the early evolutionary history of postcranial pneumaticity.

Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter.

MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

Effects of lipid environment on the conformational changes of an ABC importer.

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography.

Efficient carbon utilization is critical to the survival of microorganisms in competitive environments. To optimize energy usage, bacteria have developed an integrated control system to preferentially uptake carbohydrates that support rapid growth. The availability of a preferred carbon source, such as glucose, represses the synthesis and activities of proteins necessary for the transport and metabolism of secondary carbon sources. This regulatory phenomenon is defined as carbon catabolite repression. In enteric bacteria, the key player of carbon catabolite repression is a component of the glucose-specific phosphotransferase system, enzyme IIA (EIIA(Glc)). It is known that unphosphorylated EIIA(Glc) binds to and inhibits a variety of transporters when glucose is available. However, understanding the underlying molecular mechanism has been hindered by the complete absence of structures for any EIIA(Glc)-transporter complexes. Here we present the 3.9 Å crystal structure of Escherichia coli EIIA(Glc) in complex with the maltose transporter, an ATP-binding cassette (ABC) transporter. The structure shows that two EIIA(Glc) molecules bind to the cytoplasmic ATPase subunits, stabilizing the transporter in an inward-facing conformation and preventing the structural rearrangements necessary for ATP hydrolysis. We also show that the half-maximal inhibitory concentrations of the full-length EIIA(Glc) and an amino-terminal truncation mutant differ by 60-fold, consistent with the hypothesis that the amino-terminal region, disordered in the crystal structure, functions as a membrane anchor to increase the effective EIIA(Glc) concentration at the membrane. Together these data suggest a model of how the central regulatory protein EIIA(Glc) allosterically inhibits maltose uptake in E. coli.

EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.

In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

The ABC transporter MalFGK(2) sequesters the MalT transcription factor at the membrane in the absence of cognate substrate.

MalK, the cytoplasmic component of the maltose ABC transporter from Escherichia coli is known to control negatively the activity of MalT, the activator of the maltose regulon, through complex formation. Here we further investigate this regulatory process by monitoring MalT activity and performing fluorescence microscopy analyses under various conditions. We establish that, under physiological conditions, the molecular entity that interacts with MalT is not free MalK, but the maltose transporter, MalFGK(2) , which sequesters MalT to the membrane. Furthermore, we provide compelling evidence that the transporter's ability to bind MalT is not constitutive, but strongly diminished when MalFGK(2) is engaged in sugar transport. Notably, the outward-facing transporter, i.e. the catalytic intermediate, is ineffective in inhibiting MalT compared to the inward-facing state, i.e. the resting form. Analyses of available genetic and structural data suggest how the interaction between one inactive MalT molecule and MalFGK(2) would be sensitive to the transporter state, thereby allowing MalT release upon maltose entrance. A related mechanism may underpin signalling by other ABC transporters.