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All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Structural basis for potent neutralization of SARS-CoV-2 and role of antibody affinity maturation

SARS-CoV-2 is a betacoronavirus virus responsible for the COVID-19 pandemic. Here, we determine the X-ray crystal structure of a potent neutralizing monoclonal antibody, CV30, isolated from a patient infected with SARS-CoV-2, in complex with the receptor binding domain. The structure reveals that CV30 binds to an epitope that overlaps with the human ACE2 receptor binding motif providing a structural basis for its neutralization. CV30 also induces shedding of the S1 subunit, indicating an additional mechanism of neutralization. A germline reversion of CV30 results in a substantial reduction in both binding affinity and neutralization potential indicating the minimal somatic mutation is needed for potently neutralizing antibodies against SARS-CoV-2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structure and regulation of human epithelial cell transforming 2 protein

Epithelial cell transforming 2 (Ect2) protein activates Rho GTPases and controls cytokinesis and many other cellular processes. Dysregulation of Ect2 is associated with various cancers. Here, we report the crystal structure of human Ect2 and complementary mechanistic analyses. The data show the C-terminal PH domain of Ect2 folds back and blocks the canonical RhoA-binding site at the catalytic center of the DH domain, providing a mechanism of Ect2 autoinhibition. Ect2 is activated by binding of GTP-bound RhoA to the PH domain, which suggests an allosteric mechanism of Ect2 activation and a positive-feedback loop reinforcing RhoA signaling. Furthermore this bimodal RhoA binding of Ect2 is unusual and was confirmed with Förster resonance energy transfer (FRET) and hydrogen–deuterium exchange mass spectrometry (HDX-MS) analyses. Several recurrent cancer-associated mutations map to the catalytic and regulatory interfaces, and dysregulate Ect2 in vitro and in vivo. Together, our findings provide mechanistic insights into Ect2 regulation in normal cells and under disease conditions.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2009

Usability: Human Research Program - Space Human Factors and Habitability

The Usability project addresses the need for research in the area of metrics and methodologies used in hardware and software usability testing in order to define quantifiable and verifiable usability requirements. A usability test is a human-in-the-loop evaluation where a participant works through a realistic set of representative tasks using the hardware/software under investigation. The purpose of this research is to define metrics and methodologies for measuring and verifying usability in the aerospace domain in accordance with FY09 focus on errors, consistency, and mobility/maneuverability. Usability metrics must be predictive of success with the interfaces, must be easy to obtain and/or calculate, and must meet the intent of current Human Systems Integration Requirements (HSIR). Methodologies must work within the constraints of the aerospace domain, be cost and time efficient, and be able to be applied without extensive specialized training.

Sandor, Aniko↗
NASA NTRS2015

Human Systems Integration (HSI) Practitioner's Guide

The NASA/SP-2015-3709, Human Systems Integration (HSI) Practitioner's Guide, also known as the "HSIPG," provides a tool for implementing HSI activities within the NASA systems engineering framework. The HSIPG is written to aid the HSI practitioner engaged in a program or project (P/P), and serves as a knowledge base to allow the practitioner to step into an HSI lead or team member role for NASA missions. Additionally, this HSIPG is written to address the role of HSI in the P/P management and systems engineering communities and aid their understanding of the value added by incorporating good HSI practices into their programs and projects. Through helping to build a community of knowledgeable HSI practitioners, this document also hopes to build advocacy across the Agency for establishing strong, consistent HSI policies and practices. Human Systems Integration (HSI) has been successfully adopted (and adapted) by several federal agencies-most notably the U.S. Department of Defense (DoD) and the Nuclear Regulatory Commission (NRC)-as a methodology for reducing system life cycle costs (LCCs). These cost savings manifest themselves due to reductions in required numbers of personnel, the practice of human-centered design, decreased reliance on specialized skills for operations, shortened training time, efficient logistics and maintenance, and fewer safety-related risks and mishaps due to unintended human/system interactions. The HSI process for NASA establishes how cost savings and mission success can be realized through systems engineering. Every program or project has unique attributes. This HSIPG is not intended to provide one-size-fits-all recommendations for HSI implementation. Rather, HSI processes should be tailored to the size, scope, and goals of individual situations. The instructions and processes identified here are best used as a starting point for implementing human-centered system concepts and designs across programs and projects of varying types, including manned and unmanned, human spaceflight, aviation, robotics, and environmental science missions. The practitioner using this guide should have expertise in Systems Engineering or other disciplines involved in producing systems with anticipated human interactions. (See section 1.6 of this guide for further discussion on HSI discipline domains.) The HSIPG provides an "HSI layer" to the NASA Systems Engineering Engine (SEE), detailed in NASA Procedural Requirement (NPR) 7123.1B, NASA Systems Engineering Processes and Requirements, and further explained in NASA/SP-2007-6105, Systems Engineering Handbook (see HSIPG Table 2.2-1, NASA Documents with HSI Content, for specific references and document versions).

Zumbado, Jennifer Rochlis↗
NASA NTRS2024

Core Body Temperature Predictions Using Metabolic Energy Expenditure and Heart Rate During Simulated Extravehicular Activity

Long duration spaceflight missions will require crew to become more autonomous in conducting extravehicular activities (EVA) without direct communication with Mission Control for biomedical support. To enable such autonomy, we are developing a Crew State and Risk Model (CSRM) as a collection of key physiology domains that drive EVA crew capabilities and workloads. One model component of CSRM is human thermal regulation. In this paper, customized development of a baseline model to predict core body temperature is presented using physiology inputs of heart rate and metabolic rate. The model development dataset included a baseline study where participants (n=6, equal male and female) performed a 5-hour EVA in a two-part session while wearing a hybrid space suit simulator (HS3). The first session included an end-to-end EVA traversing 1500 meters to a geology site and traversing back to a habitat conducting geology, payload relocation, and maintenance operations every 500 meters. The second session included standalone tasks of a 2000-meter traverse followed by geology tasks. Thermal measures of core body temperature, local skin temperature, liquid cooling garment temperature, heart rate, and metabolic rate were collected through the test duration. Multiple regression was used to build a linear equation to predict core body temperature from inputs of heart rate and metabolic rate. Heat storage was calculated via predicted core body temperature plus LCG and skin temperatures. The model was tested against a dataset from a pressurized suited test (n=6, equal male and female) in the NASA Active Response Gravity Offload System (ARGOS) conducting similar end-to-end EVA and standalone tasks. Predicted error of the model against the raw test cases was 0.2±0.15 °C. The baseline prediction of core body temperature using heart rates and metabolic rates allows for simple real-time tracking from data collected in-flight to monitor crew consumables and thermal flight limits during EVA.

Bradley Hoffmann↗
NASA NTRS2006

Human Factors Engineering as a System in the Vision for Exploration

In order to accomplish NASA's Vision for Exploration, while assuring crew safety and productivity, human performance issues must be well integrated into system design from mission conception. To that end, a two-year Technology Development Project (TDP) was funded by NASA Headquarters to develop a systematic method for including the human as a system in NASA's Vision for Exploration. The specific goals of this project are to review current Human Systems Integration (HSI) standards (i.e., industry, military, NASA) and tailor them to selected NASA Exploration activities. Once the methods are proven in the selected domains, a plan will be developed to expand the effort to a wider scope of Exploration activities. The methods will be documented for inclusion in NASA-specific documents (such as the Human Systems Integration Standards, NASA-STD-3000) to be used in future space systems. The current project builds on a previous TDP dealing with Human Factors Engineering processes. That project identified the key phases of the current NASA design lifecycle, and outlined the recommended HFE activities that should be incorporated at each phase. The project also resulted in a prototype of a webbased HFE process tool that could be used to support an ideal HFE development process at NASA. This will help to augment the limited human factors resources available by providing a web-based tool that explains the importance of human factors, teaches a recommended process, and then provides the instructions, templates and examples to carry out the process steps. The HFE activities identified by the previous TDP are being tested in situ for the current effort through support to a specific NASA Exploration activity. Currently, HFE personnel are working with systems engineering personnel to identify HSI impacts for lunar exploration by facilitating the generation of system-level Concepts of Operations (ConOps). For example, medical operations scenarios have been generated for lunar habitation in order to identify HSI requirements for the lunar communications architecture. Throughout these ConOps exercises, HFE personnel are testing various tools and methodologies that have been identified in the literature. A key part of the effort is the identification of optimal processes, methods, and tools for these early development phase activities, such as ConOps, requirements development, and early conceptual design. An overview of the activities completed thus far, as well as the tools and methods investigated will be presented.

Mihriban Whitmore↗
NASA NTRS1996

Human Factors in Cabin Accident Investigations

Human factors has become an integral part of the accident investigation protocol. However, much of the investigative process remains focussed on the flight deck, airframe, and power plant systems. As a consequence, little data has been collected regarding the human factors issues within and involving the cabin during an accident. Therefore, the possibility exists that contributing factors that lie within that domain may be overlooked. The FAA Office of Accident Investigation is sponsoring a two-day workshop on cabin safety accident investigation. This course, within the workshop, will be of two hours duration and will explore relevant areas of human factors research. Specifically, the three areas of discussion are: Information transfer and resource management, fatigue and other physical stressors, and the human/machine interface. Integration of these areas will be accomplished by providing a suggested checklist of specific cabin-related human factors questions for investigators to probe following an accident.

Chute, Rebecca D.↗
NASA NTRSDate not supplied

Task Load Management in Earth Independent Medical Operations

BACKGROUND: Medical care in spaceflight carries a high task load and can easily overwhelm a small crew. Present day operations in low Earth orbit (LEO) offload most medical tasks to ground teams in mission control. This team includes dozens of flight surgeons, specialists, and engineers and supports the on-orbit crew in monitoring environmental systems, tracking medications, guiding procedures, providing expert advice, and many other tasks. However, the physical limitations of the speed of light and technical limitations of bandwidth, channel capacity, and signal processing mean that missions beyond LEO cannot rely on this level of telemedical support. The further we travel from Earth the more these tasks will fall on the shoulders of the crew and the greater the risk of task saturation to the wellbeing of the crew and the success of the mission. Exploration class space crews will need progressively more robust systems for managing task load as they progress further out in space. OVERVIEW: Medical task management systems will need to assist with two broad categories of tasks; cognitively intensive tasks and procedure execution tasks. In both cases the goal is for the systems to operate in the background with minimal human-in-the-loop intervention. To accomplish this such systems will need to be designed with careful consideration for human factors and human systems integration to maximize efficiency, minimize alarm fatigue, and avoid inadvertently increasing task loads. Finally, the key domains of space medicine tasking can be used to map present day and near future technologies to the areas where they are best suited to support and identify gaps which can be targeted for research and development. DISCUSSION: Task load is a major challenge for Earth Independent Medical Operations to overcome. It will require careful coordination between experts in a variety of fields paying attention to human factors and human systems integration as well as technical and medical expertise. If done well medical task management systems can handle many of the tasks currently run by humans in mission control and enable human crews to maintain terrestrial standards of care in the extraterrestrial environment.

Dana Levin↗
NASA NTRS2004

EVA Performance Prediction

Astronaut physical performance capabilities in micro gravity EV A or on planetary surfaces when encumbered by a life support suit and debilitated by a long exposure to micro gravity will be less than unencumbered pre flight capabilities. The big question addressed by human factors engineers is: what can the astronaut be expected to do on EVA or when we arrive at a planetary surface? A second question is: what aids to performance will be needed to enhance the human physical capability? These questions are important for a number of reasons. First it is necessary to carry out accurate planning of human physical demands to ensure that time and energy critical tasks can be carried out with confidence. Second it is important that the crew members (and their ground or planetary base monitors) have a realistic picture of their own capabilities, as excessive fatigue can lead to catastrophic failure. Third it is important to design appropriate equipment to enhance human sensory capabilities, locomotion, materials handling and manipulation. The evidence from physiological research points to musculoskeletal, cardiovascular and neurovestibular degradation during long duration exposure to micro gravity . The evidence from the biomechanics laboratory (and the Neutral Buoyancy Laboratory) points to a reduction in range of motion, strength and stamina when encumbered by a pressurized suit. The evidence from a long history of EVAs is that crewmembers are indeed restricted in their physical capabilities. There is a wealth of evidence in the literature on the causes and effects of degraded human performance in the laboratory, in sports and athletics, in industry and in other physically demanding jobs. One approach to this challenge is through biomechanical and performance modeling. Such models must be based on thorough task analysis, reliable human performance data from controlled studies, and functional extrapolations validated in analog contexts. The task analyses currently carried out for EVA activities are based more on extensive domain experience than any formal analytic structure. Conversely, physical task analysis for industrial and structured evidence from training and EV A contexts. Again on earth there is considerable evidence of human performance degradation due to encumbrance and fatigue. These industrial models generally take the form of a discounting equation. The development of performance estimates for space operations, such as timeline predictions for EVA is generally based on specific input from training activity, for example in the NBL or KC135. uniformed services tasks on earth are much more formalized. Human performance data in the space context has two sources: first there is the micro analysis of performance in structured tasks by the space physiology community and second there is the less structured evidence from training and EV A contexts.

Peacock, Brian↗
NASA NTRSDate not supplied

NSCOR for Evaluating Risk Factors and Biomarkers for Adaptation and Resilience to Spaceflight: Emotional Valence and Social Processes in ICC/ICE Environments

Space exploration class missions, such as a mission to Mars, will require optimization of human performance, adaptability, and resilience. This NASA Specialized Center of Research (NSCOR) utilizes the NIMH Research Domain Criteria (RDoC) framework to identify biological and behavioral markers of individual social adaptation and emotional resilience (as well as vulnerability) to spaceflight-relevant stressors such as living in extended isolation. The overarching goal of this NSCOR is to obtain novel information to help identify biomarkers of individuals who are resilient and/or adaptable to the stressors of isolated, confined, and controlled (ICC) and isolated, confined, and extreme (ICE) environments.A total of N=90 healthy adult astronaut surrogates are being studied in three spaceflight-analog environments: (1) n=40 healthy adults in the Isolation and Confinement Analog Research Unit (ICARUS), an ICC at the University of Pennsylvania, during 7-day missions, for a target total of 280 subject days; (2) n=32 healthy adult astronaut surrogates studied in NASA’s Human Exploration Research Analog (HERA), an ICC at Johnson Space Center during 45-day missions, for a target total of 2,112 subject days; and (3) n=18 healthy adults in the Alfred-Wegener-Institute’s Neumayer Station III, an ICE in Antarctica, during 14-month missions, for a target total of 7,560 subject days. Dr. Nindl’s Laboratory at the University of Pittsburgh is analyzing a priori selected protein biomarkers in blood, saliva, and urine. Complementary rodent models of exposure to early life stressors, confinement, and isolation are being evaluated at Dr. Hensch’s Laboratory to further validate the neurobehavioral and biological findings from the human studies.Given the inconsistency and varied definition of resilience/adaptation in the scientific literature, the NSCOR team developed a composite resilience/adaptation measure that reflects the most relevant outcomes to resilience/adaptation across psychosocial and neurobehavioral functions, as well as neurocognitive and spaceflight-relevant operational performance. To achieve this, group consensus was attained from subject matter experts to produce a rank-order of importance for each input variable. The final resilience/adaptation score included 36 variables that were collected across spaceflight analogs. As of 10/1/2021, the NSCOR project acquired data on n=27 subjects at ICARUS, n=16 at HERA, and n=18 at Neumayer. The COVID-19 pandemic delayed data acquisition at ICARUS and HERA.Among subjects studied to date, 99% of neural and neurobehavioral data (e.g., neuroimaging for structure and function, behavioral measures) as well as blood, saliva, and urine for biochemical assays have been acquired. For rodent models, Dr. Hensch’s laboratory has established biochemical and behavioral parameters reflecting confinement stress in social networks of mice for comparison to stress responses in the human spaceflight analog environments.Group social behaviors were measured with a Social Network Analysis (SNA) approach to define objective parameters associated with sociability and its plasticity by sex. This analytic approach may help identify a network of individuals who are more effective teammates or more likely to generate new social relationships. Data acquisition, biomarker assessment, and data quality control will continue through September 2022.

D F Dinges↗
NASA NTRSDate not supplied

Humans as Automation Failsafe: HAT Assistant

Humans are frequently left to “backstop” automated systems, and Human Factors specialists have argued against this for decades with, at best, partial success. What if we took a different tack... and designed to support it? The participants were involved in a recent effort to review and document cases across multiple domains where operators acted as a “failsafe” for automation, intervening in unanticipated situations to maximize success and minimize damage. We defined a “Human As Failsafe” (HAF) incident and then investigated conditions and practices making HAF success more or less likely. Analyzing these historical incidents, we suggested remediation approaches. The project also examined the legal concept of culpability (i.e., when intervention should have happened but didn’t) and proposed a state-machine-based analytic simulation to identify when HAF interventions are plausible. The panel objective will be to briefly present these concepts, but more generally to discuss designing for inevitable HAF events. This presentation with review the concept of a HAT Assistant to support multi-vehicle control of drones in a wildland firefighting context.

humans as failsafe↗
NASA NTRS2014

NASA Wearable Technology CLUSTER 2013-2014 Report

Wearable technology has the potential to revolutionize the way humans interact with one another, with information, and with the electronic systems that surround them. This change can already be seen in the dramatic increase in the availability and use of wearable health and activity monitors. These devices continuously monitor the wearer using on-­‐body sensors and wireless communication. They provide feedback that can be used to improve physical health and performance. Smart watches and head mounted displays are also receiving a great deal of commercial attention, providing immediate access to information via graphical displays, as well as additional sensing features. For the purposes of the Wearable Technology CLUSTER, wearable technology is broadly defined as any electronic sensing, human interfaces, computing, or communication that is mounted on the body. Current commercially available wearable devices primarily house electronics in rigid packaging to provide protection from flexing, moisture, and other contaminants. NASA mentors are interested in this approach, but are also interested in direct integration of electronics into clothing to enable more comfortable systems. For human spaceflight, wearable technology holds a great deal of promise for significantly improving safety, efficiency, autonomy, and research capacity for the crew in space and support personnel on the ground. Specific capabilities of interest include: Continuous biomedical monitoring for research and detection of health problems. Environmental monitoring for individual exposure assessments and alarms. Activity monitoring for responsive robotics and environments. Multi-modal caution and warning using tactile, auditory, and visual alarms. Wireless, hands-free, on-demand voice communication. Mobile, on-demand access to space vehicle and robotic displays and controls. Many technical challenges must be overcome to realize these wearable technology applications. For example, to make a wearable device that is both functional and comfortable for long duration wear, developers must strive to reduce electronic mass and volume while also addressing constraints imposed by the body attachment method. Depending on the application, the device must be placed in a location that the user can see and reach, and that provides the appropriate access to air and the wearer's skin. Limited power is available from body-­‐worn batteries and heat must be managed to prevent discomfort. If the clothing is to be washed, there are additional durability and washability hurdles that traditional electronics are not designed to address. Finally, each specific capability has unique technical challenges that will likely require unique solutions. In addition to the technical challenges, development of wearable devices is made more difficult by the diversity of skills required and the historic lack of collaboration across domains. Wearable technology development requires expertise in textiles engineering, apparel design, software and computer engineering, electronic design and manufacturing, human factors engineering, and application-­‐specific fields such as acoustics, medical devices, and sensing. Knowledge from each of these domains must be integrated to create functional and comfortable devices. For this reason, the diversity of knowledge and experience represented in the Wearable Technology is critical to overcoming the fundamental challenges in the field.

Simon, Cory↗
NASA NTRS2000

Over-Expression, Purification and Crystallization of Human Dihydrolipoamide Dehydrogenase

Dehydrolipoamide dehydrogenase (E3; dihydrolipoan-tide:NAD+ oxidoreductase, EC 1.8.1.4) is a common catalytic component found in pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and branched-chain cc-keto acid dehydrogenase complex. E3 is also a component (referred to as L protein) of the glycine cleavage system in bacterial metabolism (2). Active E3 forms a homodimer with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme. Deduced amino acids from cloned full-length human E3 gene showed a total of 509 amino acids with a leader sequence (N-terminal 35 amino acids) that is excised (mature form) during transportation of expressed E3 into mitochondria membrane. So far, three-dimensional structure of human E3 has not been reported. Our effort to achieve the elucidation of the X-ray crystal structure of human E3 will be presented. Recombinant pPROEX-1 expression vector (from GIBCO BRL Life Technologies) having the human E3 gene without leader sequence was constructed by Polymerase Chain Reaction (PCR) and subsequent ligation, and cloned in E.coli XL1-Blue by transformation. Since pPROEX-1 vector has an internal His-tag (six histidine peptide) located at the upstream region of a multicloning site, one-step affinity purification of E3 using nickelnitriloacetic acid (Ni-NTA) agarose resin, which has a strong affinity to His-tag, was feasible. Also a seven-amino-acid spacer peptide and a recombinant tobacco etch virus protease recognition site (seven amino acids peptide) found between His-tag and first amino acid of expressed E3 facilitated the cleavage of His-tag from E3 after the affinity purification. By IPTG induction, ca. 15 mg of human E3 (mature form) was obtained from 1L LB culture with overnight incubation at 25C. Over 98% of purity of E3 from one-step Ni-NTA agarose affinity purification was confirmed by SDS-PAGE analysis. For crystallization, E3 samples were prepared with and without His-tag. To minimize the aggregation of E3, apo- and holo- forms of E3s were tested, as well as a mutated E3. Dynamic light scattering measurements revealed that the E3 preparations without His-tag and substrate are highly monodispersive with regard to homodimers. Consequent crystallization trials of this E3 preparation led to single crystals of E3 grown by the vapor diffusion method. Crystals were obtained within a few days from solution containing poly (ethylene glycol) monomethyl ether 5000 as a precipitant. Autoindexing and integration of the X-ray diffraction data showed that E3 crystals belong to an orthorhombic system with unit cell parameters a-- 123. 1, b= 165.3 and c=214.3A. Further optimization of protein preparation and crystallization experiments for the structural determination will be discussed.

Hong, Y. S.↗
NASA NTRS2003

Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

Swinglel, Mark↗
NASA NTRS2003

Structural Basis for Flip-Flop Action of Thiamin Pyrophosphate-Dependent Enzymes Revealed by Human Pyruvate Dehydrogenase

Thiamin pyrophosphate (TPP), the biologically active form of vitamin BI, is a cofactor of enzymes catalyzing reactions involving the cleavage of a carbon-carbon bond adjacent to an oxo group. TPP-dependent enzymes show a common mechanism of TPP activation by: (1) forming the ionic N-H...O(sup -) hydrogen bonding between the N1' atom of the aminopirymidine ring of the coenzyme and intrinsic gamma-carboxylate group of glutamate and (2) imposing an "active" V-conformation that brings the N4' atom of the aminopirymidine to the distance required for the intramolecular C-H.. .N hydrogen bonding with the thiazolium C2 atom. Within these two hydrogen bonds that rapidly exchange protons, protonation of the N1' atom is strictly coordinated with the deprotonation of the 4' -amino group and eventually abstraction of the proton from C2. The human pyruvate dehydrogenase Elp, component of human pyruvate dehydrogenase complex, catalyzes the irreversible decarboxylation of the pyruvate followed by the reductive acetylation of the lipoyl group of dihydrolipoyl acyltransferase. Elp is alpha(sub 2)beta(sub2)-heterotetrameric with a molecular mass of I54 kDa, which has two catalytic sites, each providing TPP and magnesium ion as cofactors and each formed on the interface between the PP and PYR domains. The dynamic nonequivalence of two otherwise chemically equivalent catalytic sites has been observed and the flip-flop mechanism was suggested, according to which two active sites affect each other and in which different steps of the catalytic reaction are performed in each of the sites at any given moment. Based on specific futures of human pyruvate dehydrogenase including rigid and flexible connections between domains that bind the cofactor we propose a mechanistic model for the flip-flop action of this enzyme. We postulate that the dynamic protein environment drives the exchange of tautomers in the 4' -aminopyrimidine ring of the cofactor through a concerted shuttl-like motion of tightly connected domains. The dynamic exchange of those tautomers, in turns, is required during the reactions of pyruvate decarboxylation and reductive acetylation of lipoamide. Thus the shuttle-like motion of the domains is coordinated with the reactions of decarboxylation and acetylation, which are carried out in each of the cofactor sites resulting in a flip-flop action of the enzyme. The structure-derived mechanism of action of human pyruvate dehydrogenase may be likely common for other TPP-dependent enzymes.

Dominiak, Paulina↗
DOE OSTI2023

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1–D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 μg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1999

Differential expression of human lysyl hydroxylase genes, lysine hydroxylation, and cross-linking of type I collagen during osteoblastic differentiation in vitro

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen alpha chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of alpha chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross-linking pattern.

NASA Discipline Musculoskeletal↗