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Energy Transduction in Nitrogenase
Nitrogenase is a complicated two-component enzyme system that uses ATP binding and hydrolysis energy to achieve one of the most difficult chemical reactions in nature, the reduction of N2 to NH3. One component of the Mo-based nitrogenase system, Fe protein, delivers electrons one at a time to the second component, the catalytic MoFe protein. This process occurs through a series of synchronized events collectively called the “Fe protein cycle”. Elucidating details of the events associated with this cycle has constituted an important challenge in understanding the nitrogenase mechanism. Electron delivery is a multistep process involving three metal clusters with intra- and interprotein events. It is proposed that the first electron transfer event is a gated intraprotein transfer of one electron from the MoFe protein P-cluster to the FeMo cofactor. Measurement of the effect of osmotic pressure on the rate of this electron transfer process revealed that it is gated by protein conformational changes. This first electron transfer is activated by binding of the Fe protein containing two bound ATP molecules. The mechanism of how this protein-protein association triggers electron transfer remains unknown. The second electron transfer event is proposed to be a rapid interprotein “backfill” with transfer of one electron from the reduced Fe protein 4Fe-4S cluster to the oxidized P-cluster. In this way, electron delivery can be viewed as a case of “deficit spending”. Such a deficit-spending electron transfer process can be envisioned as a way to achieve one-direction electron flow, limiting the potential for back electron flow. Hydrolysis of two ATP molecules associated with the Fe protein occurs after the electron transfer and therefore is not used to directly drive the electron transfer. Rather, ATP hydrolysis is proposed to contribute to relaxation of the “activated” conformational state associated with the ATP form of the complex, with the free energy from ATP hydrolysis being used to pay back energy associated with component protein association and electron transfer. Release of inorganic phosphate (Pi) and protein-protein dissociation follow electron transfer and ATP hydrolysis. The ratelimiting step for the Fe protein cycle is not dissociation of the two proteins, as previously believed, but rather is release of Pi after ATP hydrolysis, which is then followed by rapid protein-protein complex dissociation. Nitrogenase is composed of two catalytic halves that do not function independently but rather exhibit anticooperative nuclear motion in which electron transfer in one-half of the complex partially inhibits electron transfer and ATP hydrolysis in the other half. Calculations indicated the existence of anticooperative interactions across the entire nitrogenase complex, suggesting a mechanism for the control of events on opposite ends of this large complex. The mechanistic necessity for this anticooperative process remains unknown. This Account presents a working model for how all of these processes work together in the nitrogenase “machine” to transduce the energy from ATP binding and hydrolysis to drive N2 reduction.
SU-E-T-269: Differential Hazard Analysis For Conventional And New Linac Acceptance Testing Procedures
Purpose: New techniques and materials have recently been developed to expedite the conventional Linac Acceptance Testing Procedure (ATP). The new ATP method uses the Electronic Portal Imaging Device (EPID) for data collection and is presented separately. This new procedure is meant to be more efficient then conventional methods. While not clinically implemented yet, a prospective risk assessment is warranted for any new techniques. The purpose of this work is to investigate the risks and establish the pros and cons between the conventional approach and the new ATP method. Methods: ATP tests that were modified and performed with the EPID were analyzed. Five domain experts (Medical Physicists) comprised the core analysis team. Ranking scales were adopted from previous publications related to TG 100. The number of failure pathways for each ATP test procedure were compared as well as the number of risk priority numbers (RPN’s) greater than 100 were compared. Results: There were fewer failure pathways with the new ATP compared to the conventional, 262 and 556, respectively. There were fewer RPN’s > 100 in the new ATP compared to the conventional, 41 and 115. Failure pathways and RPN’s > 100 for individual ATP tests on average were 2 and 3.5 times higher in the conventional ATP compared to the new, respectively. The pixel sensitivity map of the EPID was identified as a key hazard to the new ATP procedure with an RPN of 288 for verifying beam parameters. Conclusion: The significant decrease in failure pathways and RPN’s >100 for the new ATP mitigates the possibilities of a catastrophic error occurring. The Pixel Sensitivity Map determining the response and inherent characteristics of the EPID is crucial as all data and hence results are dependent on that process. Grant from Varian Medical Systems Inc.
Chemomechanical Coupling in Hexameric Protein–Protein Interfaces Harnesses Energy within V-Type ATPases
ATP synthase is the most prominent bioenergetic macromolecular motor in all life forms, utilizing the proton gradient across the cell membrane to fuel the synthesis of ATP. Notwithstanding the wealth of available biochemical and structural information inferred from years of experiments, the precise molecular mechanism whereby vacuolar (V-type) ATP synthase fulfills its biological function remains largely fragmentary. Recently, crystallographers provided the first high-resolution view of ATP activity in Enterococcus hirae V1-ATPase. Employing a combination of transition-path sampling and high-performance free-energy methods, the sequence of conformational transitions involved in a functional cycle accompanying ATP hydrolysis has been investigated in unprecedented detail over an aggregate simulation time of 65 μs. Our simulated pathways reveal that the chemical energy produced by ATP hydrolysis is harnessed via the concerted motion of the protein–protein interfaces in the V1-ring, and is nearly entirely consumed in the rotation of the central stalk. Surprisingly, in an ATPase devoid of a central stalk, the interfaces of this ring are perfectly designed for inducing ATP hydrolysis. However, in a complete V1-ATPase, the mechanical property of the central stalk is a key determinant of the rate of ATP turnover. The simulations further unveil a sequence of events, whereby unbinding of the hydrolysis product (ADP + Pi) is followed by ATP uptake, which, in turn, leads to the torque generation step and rotation of the center stalk. Molecular trajectories also bring to light multiple intermediates, two of which have been isolated in independent crystallography experiments.
Iptakalim inhibits PDGF-BB-induced human airway smooth muscle cells proliferation and migration
Chronic airway diseases are characterized by airway remodeling which is attributed partly to the proliferation and migration of airway smooth muscle cells (ASMCs). ATP-sensitive potassium (K{sub ATP}) channels have been identified in ASMCs. Mount evidence has suggested that K{sub ATP} channel openers can reduce airway hyperresponsiveness and alleviate airway remodeling. Opening K{sup +} channels triggers K{sup +} efflux, which leading to membrane hyperpolarization, preventing Ca{sup 2+}entry through closing voltage-operated Ca{sup 2+} channels. Intracellular Ca{sup 2+} is the most important regulator of muscle contraction, cell proliferation and migration. K{sup +} efflux decreases Ca{sup 2+} influx, which consequently influences ASMCs proliferation and migration. As a K{sub ATP} channel opener, iptakalim (Ipt) has been reported to restrain the proliferation of pulmonary arterial smooth muscle cells (PASMCs) involved in vascular remodeling, while little is known about its impact on ASMCs. The present study was designed to investigate the effects of Ipt on human ASMCs and the mechanisms underlying. Results obtained from cell counting kit-8 (CCK-8), flow cytometry and 5-ethynyl-2′-deoxyuridine (EdU) incorporation showed that Ipt significantly inhibited platelet-derived growth factor (PDGF)-BB-induced ASMCs proliferation. ASMCs migration induced by PDGF-BB was also suppressed by Ipt in transwell migration and scratch assay. Besides, the phosphorylation of Ca{sup 2+}/calmodulin-dependent kinase II (CaMKII), extracellular regulated protein kinases 1/2 (ERK1/2), protein kinase B (Akt), and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were as well alleviated by Ipt administration. Furthermore, we found that the inhibition of Ipt on the PDGF-BB-induced proliferation and migration in human ASMCs was blocked by glibenclamide (Gli), a selective K{sub ATP} channel antagonist. These findings provide a strong evidence to support that Ipt antagonize the proliferating and migrating effects of PDGF-BB on human ASMCs through opening K{sub ATP} channels. Altogether, our results highlighted a novel profile of Ipt as a potent option against the airway remodeling in chronic airway diseases. - Highlights: Iptakalim is a novel ATP-sensitive potassium channel opener. Iptakalim showed anti-proliferation and anti-migration effects on PDGF-BB-induced human airway smooth muscle cells. Inhibitory effects of Iptakalim could be abolished by glibenclamide, a selective K{sub ATP} channel antagonist. Inhibitory effects of Iptakalim involved the signaling pathways of CaMKII, ERK1/2 and Akt, as well as their downstream, CREB.
Structural insights into lethal contractural syndrome type 3 (LCCS3) caused by a missense mutation of PIP5Kγ
Signaling molecule phosphatidylinositol 4,5-bisphosphate is produced primarily by phosphatidylinositol 4-phosphate 5-kinase (PIP5K). PIP5K is essential for the development of the human neuronal system, which has been exemplified by a recessive genetic disorder, lethal congenital contractural syndrome type 3, caused by a single aspartate-to-asparagine mutation in the kinase domain of PIP5Kγ. So far, the exact role of this aspartate residue has yet to be elucidated. As such, we conducted structural, functional and computational studies on a zebrafish PIP5Kα variant with a mutation at the same site. Compared with the structure of the wild-type (WT) protein in the ATP-bound state, the ATP-associating glycine-rich loop of the mutant protein was severely disordered and the temperature factor of ATP was significantly higher. Both observations suggest a greater degree of disorder of the bound ATP, whereas neither the structure of the catalytic site nor the Km toward ATP was substantially affected by the mutation. Microsecond molecular dynamics simulation revealed that negative charge elimination caused by the mutation destabilized the involved hydrogen bonds and affected key electrostatic interactions in the close proximity of ATP. Taken together, our data indicated that the disease-related aspartate residue is a key node in the interaction network crucial for effective ATP binding. This work provides a paradigm of how a subtle but critical structural perturbation caused by a single mutation at the ATP-binding site abolishes the kinase activity, emphasizing that stabilizing substrate in a productive conformational state is crucial for catalysis.
A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin
Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.
Surface Analysis of 4-Aminothiophenol Adsorption at Polycrystalline Platinum Electrodes
Formation of self-assembled monolayer (SAM) of 4-aminothiophenol (4-ATP) on polycrystalline platinum electrodes has been studied by surface analysis and electrochemistry techniques. The 4-ATP monolayer was characterized by cyclic voltammetry (CV), Raman spectroscopy, reflection absorption infrared (RAIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). Cyclic voltammetry (CV) experiments give an idea about the packing quality of the monolayer. RAIR and Raman spectra for 4-ATP modified platinum electrodes showed the characteristic adsorption bands for neat 4-ATP indicating the adsorption of 4-ATP molecules on platinum surface. The adsorption on platinum was also evidenced by the presence of sulfur and nitrogen peaks by XPS survey spectra of the modified platinum electrodes. High resolution XPS studies and RAIR spectrum for platinum electrodes modified with 4-ATP indicate that molecules are sulfur-bonded to the platinum surface. The formation of S-Pt bond suggests that ATP adsorption gives up an amino terminated SAM. Thickness of the monolayer was evaluated via angle-resolved XPS (AR-XPS) analyses. Derivatization of 4-ATP SAM was performed using 16-Br hexadecanoic acid.
An Overview of the NASA Aeronautics Test Program Strategic Plan
U.S. leadership in aeronautics depends on ready access to technologically advanced, efficient, and affordable aeronautics test capabilities. These systems include major wind tunnels and propulsion test facilities and flight test capabilities. The federal government owns the majority of the major aeronautics test capabilities in the United States, primarily through the National Aeronautics and Space Administration (NASA) and the Department of Defense (DoD), however an overarching strategy for management of these national assets was needed. Therefore, in Fiscal Year (FY) 2006 NASA established the Aeronautics Test Program (ATP) as a two-pronged strategic initiative to: (1) retain and invest in NASA aeronautics test capabilities considered strategically important to the agency and the nation, and (2) establish a strong, high level partnership with the DoD Test Resources Management Center (TRMC), stewards of the DoD test and evaluation infrastructure. Since then, approximately seventy percent of the ATP budget has been directed to underpin fixed and variable costs of facility operations within its portfolio and the balance towards strategic investments in its test facilities, including maintenance and capability upgrades. Also, a strong guiding coalition was established through the National Partnership for Aeronautics Testing (NPAT), with governance by the senior leadership of NASA s Aeronautics Research Mission Directorate (ARMD) and the DoD's TRMC. As part of its strategic planning, ATP has performed or participated in many studies and analyses, including assessments of major NASA and DoD aeronautics test capabilities, test facility condition evaluations and market research. The ATP strategy has also benefitted from unpublished RAND research and analysis by Ant n et al. (2009). Together, these various studies, reports and assessments serve as a foundation for a new, five year strategic plan that will guide ATP through FY 2014. Our vision for the future is a balanced portfolio of aeronautics ground and flight test capabilities that advance U.S. leadership in aeronautics in the short and long term. Key to the ATP vision is the concept of availability, not necessarily ownership; that is, NASA does not have to own and operate all facilities that are envisioned for future aeronautics testing. However, ATP will enable access to capabilities which are needed but not owned by NASA through strategic partnerships and reliance agreements. This paper will outline the major aspects of the ATP strategic plan for achieving its mission.
Ensuring US National Aeronautics Test Capabilities
U.S. leadership in aeronautics depends on ready access to technologically advanced, efficient, and affordable aeronautics test capabilities. These systems include major wind tunnels and propulsion test facilities and flight test capabilities. The federal government owns the majority of the major aeronautics test capabilities in the United States, primarily through the National Aeronautics and Space Administration (NASA) and the Department of Defense (DoD). However, changes in the Aerospace landscape, primarily the decrease in demand for testing over the last 20 years required an overarching strategy for management of these national assets. Therefore, NASA established the Aeronautics Test Program (ATP) as a two-pronged strategic initiative to: (1) retain and invest in NASA aeronautics test capabilities considered strategically important to the agency and the nation, and (2) establish a strong, high level partnership with the DoD. Test facility utilization is a critical factor for ATP because it relies on user occupancy fees to recover a substantial part of the operations costs for its facilities. Decreasing utilization is an indicator of excess capacity and in some cases low-risk redundancy (i.e., several facilities with basically the same capability and overall low utilization). However, low utilization does not necessarily translate to lack of strategic importance. Some facilities with relatively low utilization are nonetheless vitally important because of the unique nature of the capability and the foreseeable aeronautics testing needs. Unfortunately, since its inception, the customer base for ATP has continued to shrink. Utilization of ATP wind tunnels has declined by more than 50% from the FY 2006 levels. This significant decrease in customer usage is attributable to several factors, including the overall decline in new programs and projects in the aerospace sector; the impact of computational fluid dynamics (CFD) on the design, development, and research process; and the reductions in wind tunnel testing requirements within the largest consumer of ATP wind tunnel test time, the Aeronautics Research Mission Directorate (ARMD). Retirement of the Space Shuttle Program and recent perturbations of NASA's Constellation Program will exacerbate this downward trend. Therefore it is crucial that ATP periodically revisit and determine which of its test capabilities are strategically important, which qualify as low-risk redundancies that could be put in an inactive status or closed, and address the challenges associated with both sustainment and improvements to the test capabilities that must remain active. This presentation will provide an overview of the ATP vision, mission, and goals as well as the challenges and opportunities the program is facing both today and in the future. We will discuss the strategy ATP is taking over the next five years to address the National aeronautics test capability challenges and what the program will do to capitalize on its opportunities to ensure a ready, robust and relevant portfolio of National aeronautics test capabilities.
Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle
Highlights: • Essential light chain 1 (LC1) of myosin features a unique N-terminal extension (NTE). • NTE is assumed to bind to the motor domain of myosin S1 in its ATP-bound state. • FRET distances from various positions of NTE to the motor domain are measured. • These distances strongly decrease in the S1-ADP-BeF{sub x} complex mimicking S1-AP. • Our results support the ATP-induced interaction between the NTE and motor domain. The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeF{sub x} complex (stable analog of S1{sup ∗}-AP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeF{sub x} complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.
Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28
The crystal structure of the helicase domain of the human spliceosomal DEAD-box protein Prp28 was solved by SAD. The binding of ADP and ATP by Prp28 was studied biochemically and analysed with regard to the crystal structure. The DEAD-box protein Prp28 is essential for pre-mRNA splicing as it plays a key role in the formation of an active spliceosome. Prp28 participates in the release of the U1 snRNP from the 5′-splice site during association of the U5·U4/U6 tri-snRNP, which is a crucial step in the transition from a pre-catalytic spliceosome to an activated spliceosome. Here, it is demonstrated that the purified helicase domain of human Prp28 (hPrp28ΔN) binds ADP, whereas binding of ATP and ATPase activity could not be detected. ATP binding could not be observed for purified full-length hPrp28 either, but within an assembled spliceosomal complex hPrp28 gains ATP-binding activity. In order to understand the structural basis for the ATP-binding deficiency of isolated hPrp28, the crystal structure of hPrp28ΔN was determined at 2.0 Å resolution. In the crystal the helicase domain adopts a wide-open conformation, as the two RecA-like domains are extraordinarily displaced from the productive ATPase conformation. Binding of ATP is hindered by a closed conformation of the P-loop, which occupies the space required for the γ-phosphate of ATP.
Re‐evaluating the energy balance of the many routes of carbon flow through and from photorespiration
Photorespiration is an essential process related to photosynthesis that is initiated following the oxygenation reaction catalyzed by rubisco, the initial enzyme of the Calvin–Benson–Bassham cycle. This reaction produces an inhibitory intermediate that is recycled back into the Calvin–Benson–Bassham cycle by photorespiration which requires the use of energy and the release of a portion of the carbon as CO 2 . The energy use and CO 2 release of canonical photorespiration form a foundation for biochemical models used to describe and predict leaf carbon exchange and energy use (ATP and NAPDH). The ATP and NADPH demand of canonical photorespiration is thought to be different than that of the Calvin–Benson–Bassham cycle, requiring increased flexibility in the ratio of ATP and NADPH from the light reactions. Photorespiration requires many reactions across the chloroplasts, mitochondria and peroxisomes and involves many intermediates. Growing evidence indicates that these intermediates do not all stay in photorespiration as typically assumed and instead feed into other aspects of metabolism and leave as glycine, serine, and methylene‐THF. Here we discuss how alternative flux through and from canonical photorespiration alters the ATP and NADPH requirements of metabolism following rubisco oxygenation using additional derivations of biochemical models of leaf photosynthesis and energetics. Using these new derivations, we determine that the ATP and NADPH demands of photorespiration are highly sensitive to alternative flux in ways that fundamentally changes how photorespiration contributes to the ratio of total ATP and NADPH demand. Specifically, alternative flows of carbon through photorespiration could reduce ATP and NADPH demand ratio to values below what is produced from linear electron transport.
Plasmodesmal-mediated cell-to-cell transport in wheat roots is modulated by anaerobic stress
Cell-to-cell transport of small molecules and ions occurs in plants through plasmodesmata. Plant roots are frequently subjected to localized anaerobic stress, with a resultant decrease in ATP. In order to determine the effect of this stress on plasmodesmal transport, fluorescent dyes of increasing molecular weight (0.46 to 1OkDa) were injected into epidermal and cortical cells of 3-day-old wheat roots, and their movement into neighboring cells was determined by fluorescence microscopy. Anaerobiosis was generated by N2 gas or simulated by the presence of sodium azide, both of which reduced the ATP levels in the tissue by over 80%. In the absence of such stress, the upper limit for movement, or size exclusion limit (SEL), of cortical plasmodesmata was <1 kDa. The ATP analogue TNP-ADP (mw 681) moved across the plasmodesmata of unstressed roots, indicating that plasmodesmata may be conduits for nucleotide (ATP and ADP) exchange between cells. Upon imposition of stress, the SEL rose to between 5 and 10 kDa. This response of plasmodesmata to a decrease in the level of ATP suggests that they are constricted by an ATP-dependent process so as to maintain a restricted SEL. When roots are subjected to anaerobic stress, an increase in SEL may permit enhanced delivery of sugars to the affected cells of the root where anaerobic respiration could regenerate the needed ATP.
Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport
Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.
Characterization of a novel domain ‘GATE’ in the ABC protein DrrA and its role in drug efflux by the DrrAB complex
A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homolog MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis. - Highlights: • A novel domain ‘GATE’ is identified in the ABC protein DrrA. • GATE shows high sequence and structural conservation among diverse ABC proteins. • GATE is located outside of the previously studied ATP binding and hydrolysis motifs. • Conserved GATE residues are critical for stability of DrrAB and for ATP catalysis.