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Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion–integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.
Lee Makowski; William Olson-Sidford; John W.-Weisel. Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein. Viruses 2021, 13, 146 .
AMA StyleLee Makowski, William Olson-Sidford, John W.-Weisel. Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein. Viruses. 2021; 13 (2):146.
Chicago/Turabian StyleLee Makowski; William Olson-Sidford; John W.-Weisel. 2021. "Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein." Viruses 13, no. 2: 146.
Ras dimerization is critical for Raf activation, yet Ras alone does not dimerize. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics (MD) simulations show robust allosteric connections linking the two Raf-RBD D113 residues, located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway.
Morgan Packer; Jillian A. Parker; Jean K. Chung; Zhenlu Li; Young Kwang Lee; Trinity Cookis; Hugo Guterres; Steven Alvarez; Amin Hossain; Daniel P. Donnelly; Jeffrey N. Agar; Lee Makowski; Matthias Buck; Jay T. Groves; Carla Mattos. Raf promotes dimerization of the Ras G-domain with increased allosteric connections. 2020, 1 .
AMA StyleMorgan Packer, Jillian A. Parker, Jean K. Chung, Zhenlu Li, Young Kwang Lee, Trinity Cookis, Hugo Guterres, Steven Alvarez, Amin Hossain, Daniel P. Donnelly, Jeffrey N. Agar, Lee Makowski, Matthias Buck, Jay T. Groves, Carla Mattos. Raf promotes dimerization of the Ras G-domain with increased allosteric connections. . 2020; ():1.
Chicago/Turabian StyleMorgan Packer; Jillian A. Parker; Jean K. Chung; Zhenlu Li; Young Kwang Lee; Trinity Cookis; Hugo Guterres; Steven Alvarez; Amin Hossain; Daniel P. Donnelly; Jeffrey N. Agar; Lee Makowski; Matthias Buck; Jay T. Groves; Carla Mattos. 2020. "Raf promotes dimerization of the Ras G-domain with increased allosteric connections." , no. : 1.
Amyloid fibrils represent one of the defining features of Alzheimer's disease (AD). They are made up of protofilaments comprised of Aβ peptides that are held together with extraordinary stability by a network of tight steric zippers and axial hydrogen bonds. This review explores the hypothesis that the peptide conformation within a protofilament represents the physical embodiment of a 'strain' of AD. Evidence suggests that within a single strain the fold of individual peptides is invariant. But the fibrils are capable of structural polymorphism that includes variation in the arrangement of protofilaments into fibrils; the pitch of the resultant fibrils and the higher order organization of the plaques into which they aggregate. These intra-strain polymorphisms are separated by low-energy barriers allowing multiple configurations to coexist within a single preparation or tissue. Clinical presentation of different strains may be determined by variation in the way different protofilament structures generate the relevant toxic species, be they monomers, oligomers or higher order structures. Evidence reviewed here is consistent with a model in which disease progression is concomitant with a gradual, progressive annealing of amyloid fibrils from benign, loosely packed structures into dense neurotoxic aggregates. This model challenges the commonly held hypothesis that oligomers of Aβ peptides are the only active proximate species in neurodegeneration. However, the data do not implicate fibrils themselves. Rather it casts suspicion on larger-scale supramolecular aggregates as toxic agents. Electron tomography of amyloid plaques in situ strongly suggests that formation of amyloid aggregates results in perturbation of the cellular membrane integrity, warranting further investigation of this as a potential mode of neurotoxicity. If dense, supramolecular amyloid aggregates prove to be important agents of neurodegeneration in AD, this model may also have relevance to other forms of amyloidoses.
Lee Makowski. The Structural Basis of Amyloid Strains in Alzheimer’s Disease. ACS Biomaterials Science & Engineering 2019, 6, 2498 -2505.
AMA StyleLee Makowski. The Structural Basis of Amyloid Strains in Alzheimer’s Disease. ACS Biomaterials Science & Engineering. 2019; 6 (5):2498-2505.
Chicago/Turabian StyleLee Makowski. 2019. "The Structural Basis of Amyloid Strains in Alzheimer’s Disease." ACS Biomaterials Science & Engineering 6, no. 5: 2498-2505.
Deposits of Aβ peptides (plaques) and tau protein (neurofibrillary tangles (NFTs)) are ubiquitous features of brain tissue in Alzheimer’s disease. Their contribution to disease etiology remains controversial. The molecular-to-nano-scale organization of fibrillar species in these protein aggregates remains uncertain, but may contain clues as to the contributions of these structures to disease. Whether or not all plaques are the same structure, and all tangles are the same, has implications for current hypotheses about polymorphic templated misfolding of their constituent proteins, Aβ and tau. Here we use x-ray microdiffraction in the small-angle regime (SAXS) to probe the molecular organization of these deposits. Using unstained histological sections of human brain tissue, we demonstrate that SAXS can characterize Aβ fibrils and tau filaments in situ. Aβ fibrils have a cross-sectional radius of gyration (Rxc) of ~45 Å, and larger (Rxc >150 Å) aggregates appear to represent Aβ fibrils that have coalesced side-to-side with one another to create fibrillar bundles or macrofibrillar aggregates. Tau fibrils exhibit an Rxc of ~55 Å with little sign of coalescence into larger structure. The in situ mapping of these structures revealed subtle variation in Aβ structure across different brain areas and different cases.
Biel Roig Solvas; Bradley T. Hyman; Lee Makowski. In situ SAXS of protein deposits in Alzheimer’s disease. bioRxiv 2019, 868273 .
AMA StyleBiel Roig Solvas, Bradley T. Hyman, Lee Makowski. In situ SAXS of protein deposits in Alzheimer’s disease. bioRxiv. 2019; ():868273.
Chicago/Turabian StyleBiel Roig Solvas; Bradley T. Hyman; Lee Makowski. 2019. "In situ SAXS of protein deposits in Alzheimer’s disease." bioRxiv , no. : 868273.
Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.
Rui Hu; João V. Rodrigues; Pradeep Waduge; Hirohito Yamazaki; Benjamin Cressiot; Yasmin Chishti; Lee Makowski; Dapeng Yu; Eugene Shakhnovich; Qing Zhao; Meni Wanunu. Differential Enzyme Flexibility Probed Using Solid-State Nanopores. ACS Nano 2018, 12, 4494 -4502.
AMA StyleRui Hu, João V. Rodrigues, Pradeep Waduge, Hirohito Yamazaki, Benjamin Cressiot, Yasmin Chishti, Lee Makowski, Dapeng Yu, Eugene Shakhnovich, Qing Zhao, Meni Wanunu. Differential Enzyme Flexibility Probed Using Solid-State Nanopores. ACS Nano. 2018; 12 (5):4494-4502.
Chicago/Turabian StyleRui Hu; João V. Rodrigues; Pradeep Waduge; Hirohito Yamazaki; Benjamin Cressiot; Yasmin Chishti; Lee Makowski; Dapeng Yu; Eugene Shakhnovich; Qing Zhao; Meni Wanunu. 2018. "Differential Enzyme Flexibility Probed Using Solid-State Nanopores." ACS Nano 12, no. 5: 4494-4502.
The cover image, by Yujing Wang and Lee Makowski, is based on the Research Article Fine structure of conformational ensembles in adenylate kinase, DOI: 10.1002/prot.25443.
Yujing Wang; Lee Makowski. Cover Image, Volume 86, Issue 3. Proteins: Structure, Function, and Bioinformatics 2018, 86, C1 -C1.
AMA StyleYujing Wang, Lee Makowski. Cover Image, Volume 86, Issue 3. Proteins: Structure, Function, and Bioinformatics. 2018; 86 (3):C1-C1.
Chicago/Turabian StyleYujing Wang; Lee Makowski. 2018. "Cover Image, Volume 86, Issue 3." Proteins: Structure, Function, and Bioinformatics 86, no. 3: C1-C1.
Adenylate kinase (ADK) catalyzes the reversible Mg2+-dependent phosphoryl transfer reaction Mg2++2ADP ↔Mg2++ATP + AMP in essential cellular systems. This reaction is a major player in cellular energy homeostasis and the isoform network of ADK plays an important role in AMP metabolic signaling circuits. ADK has three domains, the LID, NMP and CORE domains, that undergo large conformational rearrangements during ADK's catalytic cycle. In spite of extensive experimental and computational studies, details of the conformational pathway from open to closed forms remain uncertain. In this paper we explore this pathway using coarse-grained molecular dynamics (MD) trajectories of ADK calculated by GROMACS using a SMOG model and classify the conformations within the resultant trajectories by K-means clustering. ADK conformations segregate naturally into open; intermediate; and closed forms with long-term residence in the intermediate state. Structural clustering divides the intermediate conformation into three sub-states that are distinguished from one another on the basis of differences in both structure and dynamics. These distinctions are defined on the basis of a number of different metrics including radius of gyration, dihedral angle fluctuation, and fluctuations of interatomic pair distances. Furthermore, differences in the sub-states appear to correspond to the distinct ways each sub-state contributes to the molecular mechanism of catalysis: One sub-state acts as a gate-way to the open conformation; one sub-state a gate-way to the closed conformation. A third intermediate sub-state appears to represent a metastable off-pathway structure that is nevertheless frequently visited during the passage from open to closed state.
Yujing Wang; Lee Makowski. Fine structure of conformational ensembles in adenylate kinase. Proteins: Structure, Function, and Bioinformatics 2017, 86, 332 -343.
AMA StyleYujing Wang, Lee Makowski. Fine structure of conformational ensembles in adenylate kinase. Proteins: Structure, Function, and Bioinformatics. 2017; 86 (3):332-343.
Chicago/Turabian StyleYujing Wang; Lee Makowski. 2017. "Fine structure of conformational ensembles in adenylate kinase." Proteins: Structure, Function, and Bioinformatics 86, no. 3: 332-343.
Extending collection of x-ray solution scattering data into the wide-angle regime (WAXS) can provide information not readily extracted from small angle (SAXS) data. It is possible to accurately predict WAXS scattering on the basis of atomic coordinate sets and thus use it as a means of testing molecular models constructed on the basis of crystallography, molecular dynamics (MD), cryo-electron microscopy or ab initio modeling. WAXS data may provide insights into the secondary, tertiary and quaternary structural organization of macromolecules. It can provide information on protein folding and unfolding beyond that attainable from SAXS data. It is particularly sensitive to structural fluctuations in macromolecules and can be used to generate information about the conformational make up of ensembles of structures co-existing in solution. Novel approaches to modeling of structural fluctuations can provide information on the spatial extent of large-scale structural fluctuations that are difficult to obtain by other means. Direct comparison with the results of MD simulations are becoming possible. Because it is particularly sensitive to small changes in structure and flexibility it provides unique capabilities for the screening of ligand libraries for detection of functional interactions. WAXS thereby provides an important extension of SAXS that can generate structural and dynamic information complementary to that obtainable by other biophysical techniques.
Yujing Wang; Hao Zhou; Emre Onuk; John Badger; Lee Makowski. What Can We Learn from Wide-Angle Solution Scattering? Single Molecule and Single Cell Sequencing 2017, 1009, 131 -147.
AMA StyleYujing Wang, Hao Zhou, Emre Onuk, John Badger, Lee Makowski. What Can We Learn from Wide-Angle Solution Scattering? Single Molecule and Single Cell Sequencing. 2017; 1009 ():131-147.
Chicago/Turabian StyleYujing Wang; Hao Zhou; Emre Onuk; John Badger; Lee Makowski. 2017. "What Can We Learn from Wide-Angle Solution Scattering?" Single Molecule and Single Cell Sequencing 1009, no. : 131-147.
Crystal structures of adenylate kinase (AdK) from Escherichia coli capture two states: an “open” conformation (apo) obtained in the absence of ligands and a “closed” conformation in which ligands are bound. Other AdK crystal structures suggest intermediate conformations that may lie on the transition pathway between these two states. To characterize the transition from open to closed states in solution, X-ray solution scattering data were collected from AdK in the apo form and with progressively increasing concentrations of five different ligands. Scattering data from apo AdK are consistent with scattering predicted from the crystal structure of AdK in the open conformation. In contrast, data from AdK samples saturated with Ap5A do not agree with that calculated from AdK in the closed conformation. Using cluster analysis of available structures, we selected representative structures in five conformational states: open, partially open, intermediate, partially closed, and closed. We used these structures to estimate the relative abundances of these states for each experimental condition. X-ray solution scattering data obtained from AdK with AMP are dominated by scattering from AdK in the open conformation. For AdK in the presence of high concentrations of ATP and ADP, the conformational ensemble shifts to a mixture of partially open and closed states. Even when AdK is saturated with Ap5A, a significant proportion of AdK remains in a partially open conformation. These results are consistent with an induced-fit model in which the transition of AdK from an open state to a closed state is initiated by ATP binding.
Emre Onuk; John Badger; Yu Jing Wang; Jaydeep Bardhan; Yasmin Chishti; Murat Akcakaya; Dana H. Brooks; Deniz Erdogmus; David D. L. Minh; Lee Makowski. Effects of Catalytic Action and Ligand Binding on Conformational Ensembles of Adenylate Kinase. Biochemistry 2017, 56, 4559 -4567.
AMA StyleEmre Onuk, John Badger, Yu Jing Wang, Jaydeep Bardhan, Yasmin Chishti, Murat Akcakaya, Dana H. Brooks, Deniz Erdogmus, David D. L. Minh, Lee Makowski. Effects of Catalytic Action and Ligand Binding on Conformational Ensembles of Adenylate Kinase. Biochemistry. 2017; 56 (34):4559-4567.
Chicago/Turabian StyleEmre Onuk; John Badger; Yu Jing Wang; Jaydeep Bardhan; Yasmin Chishti; Murat Akcakaya; Dana H. Brooks; Deniz Erdogmus; David D. L. Minh; Lee Makowski. 2017. "Effects of Catalytic Action and Ligand Binding on Conformational Ensembles of Adenylate Kinase." Biochemistry 56, no. 34: 4559-4567.
Yujing Wang; Emre Onuk; Lee Makowski. The Mechanism of Population Shifting among Transition States of Adenylate Kinase. Biophysical Journal 2015, 108, 59a .
AMA StyleYujing Wang, Emre Onuk, Lee Makowski. The Mechanism of Population Shifting among Transition States of Adenylate Kinase. Biophysical Journal. 2015; 108 (2):59a.
Chicago/Turabian StyleYujing Wang; Emre Onuk; Lee Makowski. 2015. "The Mechanism of Population Shifting among Transition States of Adenylate Kinase." Biophysical Journal 108, no. 2: 59a.