Publications by authors named "Madeleine Strickland"

20 Publications

  • Page 1 of 1

Novel Tsg101 Binding Partners Regulate Viral L Domain Trafficking.

Viruses 2021 06 15;13(6). Epub 2021 Jun 15.

Department of Microbiology & Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.

Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.
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http://dx.doi.org/10.3390/v13061147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8232796PMC
June 2021

Author Correction: Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Nat Struct Mol Biol 2020 Sep;27(9):870

Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41594-020-0498-1DOI Listing
September 2020

Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

Nat Struct Mol Biol 2020 09 3;27(9):802-813. Epub 2020 Aug 3.

Cell Biology and Biophysics Unit, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA.

Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.
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http://dx.doi.org/10.1038/s41594-020-0462-0DOI Listing
September 2020

RNA Binding Suppresses Tsg101 Recognition of Ub-Modified Gag and Facilitates Recruitment to the Plasma Membrane.

Viruses 2020 04 15;12(4). Epub 2020 Apr 15.

Department of Microbiology & Immunology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794-5222, USA.

The ESCRT-I factor Tsg101 is essential for sorting endocytic cargo and is exploited by viral pathogens to facilitate egress from cells. Both the nucleocapsid (NC) domain and p6 domain in HIV-1 Gag contribute to recruitment of the protein. However, the role of NC is unclear when the P(S/T)AP motif in p6 is intact, as the motif recruits Tsg101 directly. The zinc fingers in NC bind RNA and membrane and are critical for budding. Tsg101 can substitute for the distal ZnF (ZnF2) and rescue budding of a mutant made defective by deletion of this element. Here, we report that the ubiquitin (Ub) E2 variant (UEV) domain in Tsg101 binds tRNA in vitro. We confirmed that Tsg101 can substitute for ZnF2 when provided at the viral assembly site as a chimeric Gag-Tsg101 protein (Gag-ΔZnF2-Tsg101) and rescue budding. The UEV was not required in this context; however, mutation of the RNA binding determinants in UEV prevented Tsg101 recruitment from the cell interior when Gag and Tsg101 were co-expressed. The same Tsg101 mutations increased recognition of Gag-Ub, suggesting that tRNA and Ub compete for binding sites. This study identifies a novel Tsg101 binding partner that may contribute to its function in recognition of Ub-modified cargo.
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http://dx.doi.org/10.3390/v12040447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232412PMC
April 2020

Selective Targeting of Virus Replication by Proton Pump Inhibitors.

Sci Rep 2020 03 4;10(1):4003. Epub 2020 Mar 4.

Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY, 11794-5222, USA.

Two proton pump inhibitors, tenatoprazole and esomeprazole, were previously shown to inhibit HIV-1 egress by blocking the interaction between Tsg101, a member of the ESCRT-I complex, and ubiquitin. Here, we deepen our understanding of prazole budding inhibition by studying a range of viruses in the presence of tenatoprazole. Furthermore, we investigate the relationship between the chemistry of prodrug activation and HIV-1 inhibition for diverse prazoles currently on the market. We report that tenatoprazole is capable of inhibiting the replication of members of the enveloped filo, alpha, and herpes virus families but not the flavivirus group and not the non-enveloped poliovirus. Another key finding is that prazole prodrugs must be activated inside the cell, while their rate of activation in vitro correlated to their efficacy in cells. Our study lays the groundwork for future efforts to repurpose prazole-based compounds as antivirals that are both broad-spectrum and selective in nature.
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http://dx.doi.org/10.1038/s41598-020-60544-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055211PMC
March 2020

Comparison of Solution Properties of Polymethylated DOTA-like Lanthanide Complexes with Opposite Chirality of the Pendant Arms.

Inorg Chem 2019 Dec 12;58(23):15788-15800. Epub 2019 Nov 12.

Imaging Probe Development Center, National Heart, Lung, and Blood Institute , National Institutes of Health , Rockville , Maryland 20850 , United States.

Polymethylated lanthanide 44-M4DOTMA complexes, bearing the ring methyl groups oriented in the position and the arm methyl groups in the configuration, exist exclusively as the SAP [Λ(δδδδ)] isomer in solution throughout the lanthanide series. This observation is in contrast to Ln-8-M4DOTMA, which was recently reported to adopt the SAP [Λ(δδδδ)] isomer in the early lanthanides, while the late lanthanides adopt the TSAP [Δ(δδδδ)] isomer. The methyl groups on the ring and the arm are both oriented in the configuration for Ln-8-M4DOTMA ( 2016 , 45 , 4673 - 4687 , DOI: 10.1039/C5DT03210E ). Quantum chemical calculations for Pr- and Yb-44-M4DOTMA indicate that the SAP isomer is significantly more stable. The luminescence profiles of Eu-8-M4DOTMA and Eu-44-M4DOTMA showed similar profiles signifying identical coordination environments. The hydration state, , of the Eu(III) complexes was = 0.91-0.95, while Tb-8-M4DOTMA had = 0.86. A much lower value was obtained for Tb-44-M4DOTMA ( = 0.67), which indicates an elongation of the Ln-Ow bond. At 400 MHz, the relaxivity of Gd-8-M4DOTMA is 5.1 ± 0.1 mM s and 3.9 ± 0.1 mM s at 25 and 37 °C, respectively, whereas the relaxivity of Gd-44-M4DOTMA is 4.6 ± 0.1 mM s at 25 °C and 3.6 ± 0.1 mM s at 37 °C. At 45 MHz, the relaxivity of Gd-8-M4DOTMA is 5.4 ± 0.1 mM s, and the relaxivity of Gd-44-M4DOTMA is 4.5 ± 0.1 mM s at 25 °C. The temperature dependence of the O NMR transverse relaxation rate of the Gd complexes revealed a 7-fold increase in the bound water residence lifetime of Gd-8-M4DOTMA (1/ = τ = 9.0 ± 0.5 ns and 1/ = τ = 60 ± 3 ns). The Pr(III) complex of 8-M4DOTMA crystallized as TSAP isomer with an apical water. The presence of the apical water for the TSAP of Pr-8-M4DOTMA was further confirmed with the observation that the fluoride ion replaces the bound water from the TSAP isomer of Pr-8-M4DOTMA. This was shown by the disappearance of the TSAP peaks and appearance of a new set of less shifted resonances, which exchange with the SAP isomer as confirmed by NMR exchange spectroscopy (EXSY).
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http://dx.doi.org/10.1021/acs.inorgchem.9b02049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371676PMC
December 2019

Model of a Kinetically Driven Crosstalk between Paralogous Protein Encounter Complexes.

Biophys J 2019 11 2;117(9):1655-1665. Epub 2019 Oct 2.

Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, Bethesda, Maryland. Electronic address:

Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these "futile" encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.
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http://dx.doi.org/10.1016/j.bpj.2019.09.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838881PMC
November 2019

Potential Regulatory Role of Competitive Encounter Complexes in Paralogous Phosphotransferase Systems.

J Mol Biol 2019 05 6;431(12):2331-2342. Epub 2019 May 6.

Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS) and one for nitrogen regulation (PTS), that utilize proteins enzyme I (EI) and HPr, and enzyme I (EI) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI. The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.
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http://dx.doi.org/10.1016/j.jmb.2019.04.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554058PMC
May 2019

Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein.

J Am Chem Soc 2019 01 22;141(4):1430-1434. Epub 2019 Jan 22.

Laboratory of Structural Biophysics, Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute , National Institutes of Health , Bethesda , Maryland 20892 , United States.

NMR has provided a wealth of structural and dynamical information for RNA molecules of up to ∼50 nucleotides, but its application to larger RNAs has been hampered in part by difficulties establishing global structural features. A potential solution involves measurement of NMR perturbations after site-specific paramagnetic labeling. Although the approach works well for proteins, the inability to place the label at specific sites has prevented its application to larger RNAs transcribed in vitro. Here, we present a strategy in which RNA loop residues are modified to promote binding to a paramagnetically tagged reporter protein. Lanthanide-induced pseudocontact shifts are demonstrated for a 232-nucleotide RNA bound to tagged derivatives of the spliceosomal U1A RNA-binding domain. Further, the method is validated with a 36-nucleotide RNA for which measured NMR values agreed with predictions based on the previously known protein and RNA structures. The ability to readily insert U1A binding sites into ubiquitous hairpin and/or loop structures should make this approach broadly applicable for the atomic-level study of large RNAs.
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http://dx.doi.org/10.1021/jacs.8b11384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572783PMC
January 2019

Solvent saturation transfer to proteins (SSTP) for structural and functional characterization of proteins.

J Biomol NMR 2018 01 30;70(1):11-20. Epub 2017 Nov 30.

Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein's correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer. Here, we demonstrate the utility of SSTP in structure calculations of two proteins, TSG101 and ubiquitin. The observed SSTP was found to be directly proportional to solvent accessibility. Since SSTP does not involve the direct excitation of water, which compromises the analysis of protein protons entangled in the breadth of the water resonance, it has an advantage over conventional water-based magnetization transfers. Inclusion of structural restraints derived from SSTP improved both the precision and accuracy of the final protein structures in comparison to those determined by traditional approaches, when using minimal amounts of additional structural data. Furthermore, we show that SSTP can detect weak protein-protein interactions which are unobservable by chemical shift perturbations.
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http://dx.doi.org/10.1007/s10858-017-0151-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5820130PMC
January 2018

Tsg101 chaperone function revealed by HIV-1 assembly inhibitors.

Nat Commun 2017 11 9;8(1):1391. Epub 2017 Nov 9.

Department of Molecular Genetics & Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY, 11794-5222, USA.

HIV-1 replication requires Tsg101, a component of cellular endosomal sorting complex required for transport (ESCRT) machinery. Tsg101 possesses an ubiquitin (Ub) E2 variant (UEV) domain with a pocket that can bind PT/SAP motifs and another pocket that can bind Ub. The PTAP motif in the viral structural precursor polyprotein, Gag, allows the recruitment of Tsg101 and other ESCRTs to virus assembly sites where they mediate budding. It is not known how or even whether the UEV Ub binding function contributes to virus production. Here, we report that disruption of UEV Ub binding by commonly used drugs arrests assembly at an early step distinct from the late stage involving PTAP binding disruption. NMR reveals that the drugs form a covalent adduct near the Ub-binding pocket leading to the disruption of Ub, but not PTAP binding. We conclude that the Ub-binding pocket has a chaperone function involved in bud initiation.
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http://dx.doi.org/10.1038/s41467-017-01426-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680296PMC
November 2017

Structure of the NPr:EIN Complex: Mechanism for Specificity in Paralogous Phosphotransferase Systems.

Structure 2016 12 10;24(12):2127-2137. Epub 2016 Nov 10.

Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EI and NPr:EI, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EI interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EI complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EI (EIN), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.
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http://dx.doi.org/10.1016/j.str.2016.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5143221PMC
December 2016

Characterizing the magnetic susceptibility tensor of lanthanide-containing polymethylated-DOTA complexes.

J Biomol NMR 2016 10 22;66(2):125-139. Epub 2016 Sep 22.

Laboratory of Molecular Biophysics, Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Lanthanide complexes based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) cage are commonly used as phase contrast agents in magnetic resonance imaging, but can also be utilized in structural NMR applications due to their ability to induce either paramagnetic relaxation enhancement or a pseudocontact shift (PCS) depending on the choice of the lanthanide. The size and sign of the PCS for any given atom is determined by its coordinates relative to the metal center, and the characteristics of the lanthanide's magnetic susceptibility tensor. Using a polymethylated DOTA tag (Ln-M8-SPy) conjugated to ubiquitin, we calculated the position of the metal center and characterized the susceptibility tensor for a number of lanthanides (dysprosium, thulium, and ytterbium) under a range of pH and temperature conditions. We found that there was a difference in temperature sensitivity for each of the complexes studied, which depended on the size of the lanthanide ion as well as the isomeric state of the cage. Using O-NMR, we confirmed that the temperature sensitivity of the compounds was enhanced by the presence of an apically bound water molecule. Since amide-containing lanthanide complexes are known to be pH sensitive and can be used as probes of physiological pH, we also investigated the effect of pH on the Ln-M8-SPy susceptibility tensor, but we found that the changes in this pH range (5.0-7.4) were not significant.
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http://dx.doi.org/10.1007/s10858-016-0061-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628275PMC
October 2016

Increasing the Chemical-Shift Dispersion of Unstructured Proteins with a Covalent Lanthanide Shift Reagent.

Angew Chem Int Ed Engl 2016 11 20;55(47):14847-14851. Epub 2016 Oct 20.

Center for Integrated Protein Science Munich, Technische Universität München, Department of Chemistry, Lichtenbergstraße 4, 85748, Garching, Germany.

The study of intrinsically disordered proteins (IDPs) by NMR often suffers from highly overlapped resonances that prevent unambiguous chemical-shift assignments, and data analysis that relies on well-separated resonances. We present a covalent paramagnetic lanthanide-binding tag (LBT) for increasing the chemical-shift dispersion and facilitating the chemical-shift assignment of challenging, repeat-containing IDPs. Linkage of the DOTA-based LBT to a cysteine residue induces pseudo-contact shifts (PCS) for resonances more than 20 residues from the spin-labeling site. This leads to increased chemical-shift dispersion and decreased signal overlap, thereby greatly facilitating chemical-shift assignment. This approach is applicable to IDPs of varying sizes and complexity, and is particularly helpful for repeat-containing IDPs and low-complexity regions. This results in improved efficiency for IDP analysis and binding studies.
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http://dx.doi.org/10.1002/anie.201607261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5146990PMC
November 2016

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist.

Proc Natl Acad Sci U S A 2016 May 2;113(20):E2766-75. Epub 2016 May 2.

Tumour Growth Control Group, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom;

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.
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http://dx.doi.org/10.1073/pnas.1513023113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878476PMC
May 2016

Analysis of the isomer ratios of polymethylated-DOTA complexes and the implications on protein structural studies.

Dalton Trans 2016 Mar 9;45(11):4673-87. Epub 2016 Feb 9.

Imaging Probe Development Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville, MD 20850, USA.

A rigidified and symmetrical polymethylated 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ligand bearing four SSSS methyl groups in both the tetraaza ring and the acetate arms (SSSS-SSSS-M4DOTMA) was prepared. The isomer ratio of SSSS-SSSS-M4DOTMA complexed with a series of lanthanide ions was carefully investigated using RP-HPLC and NMR. A square antiprismatic (SAP) configuration was exclusively observed for the early lanthanides, while the twisted square antiprismatic (TSAP) geometry was preferred as the lanthanide ion size decreases. The late lanthanides preferentially adopted the TSAP geometry. One of the pendant arms was modified with a pyridyl disulfide group (SSSS-SSSS-M8SPy) for cysteine attachment and displayed a similar isomer trend as the parent compound, Ln-SSSS-SSSS-M4DOTMA. Covalent attachment to the ubiquitin S57C mutant showed resonances whose intensities are in agreement with the isomeric population observed by RP-HPLC. Furthermore, the NOE experiments combined with quantum chemical calculations have unequivocally demonstrated that the SAP of Pr-SSSS-SSSS-M4DOTMA and Pr-SSSS-SSSS-M8SPy, as well as the TSAP of Yb-SSSS-SSSS-M8SPy are more stable than their corresponding isomers.
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http://dx.doi.org/10.1039/c5dt03210eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807635PMC
March 2016

Exploiting image registration for automated resonance assignment in NMR.

J Biomol NMR 2015 Jun 1;62(2):143-56. Epub 2015 Apr 1.

Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Building 50, Room 3503, Bethesda, MD, 20892, USA.

Analysis of protein NMR data involves the assignment of resonance peaks in a number of multidimensional data sets. To establish resonance assignment a three-dimensional search is used to match a pair of common variables, such as chemical shifts of the same spin system, in different NMR spectra. We show that by displaying the variables to be compared in two-dimensional plots the process can be simplified. Moreover, by utilizing a fast Fourier transform cross-correlation algorithm, more common to the field of image registration or pattern matching, we can automate this process. Here, we use sequential NMR backbone assignment as an example to show that the combination of correlation plots and segmented pattern matching establishes fast backbone assignment in fifteen proteins of varying sizes. For example, the 265-residue RalBP1 protein was 95.4% correctly assigned in 10 s. The same concept can be applied to any multidimensional NMR data set where analysis comprises the comparison of two variables. This modular and robust approach offers high efficiency with excellent computational scalability and could be easily incorporated into existing assignment software.
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http://dx.doi.org/10.1007/s10858-015-9926-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452424PMC
June 2015

The conformational changes induced by ubiquinone binding in the Na+-pumping NADH:ubiquinone oxidoreductase (Na+-NQR) are kinetically controlled by conserved glycines 140 and 141 of the NqrB subunit.

J Biol Chem 2014 Aug 8;289(34):23723-33. Epub 2014 Jul 8.

From the Laboratoire de Bioélectrochimie et Spectroscopie, UMR 7140, CNRS Université de Strasbourg, Strasbourg, France, 67000 and

Na(+)-pumping NADH:ubiquinone oxidoreductase (Na(+)-NQR) is responsible for maintaining a sodium gradient across the inner bacterial membrane. This respiratory enzyme, which couples sodium pumping to the electron transfer between NADH and ubiquinone, is not present in eukaryotes and as such could be a target for antibiotics. In this paper it is shown that the site of ubiquinone reduction is conformationally coupled to the NqrB subunit, which also hosts the final cofactor in the electron transport chain, riboflavin. Previous work showed that mutations in conserved NqrB glycine residues 140 and 141 affect ubiquinone reduction and the proper functioning of the sodium pump. Surprisingly, these mutants did not affect the dissociation constant of ubiquinone or its analog HQNO (2-n-heptyl-4-hydroxyquinoline N-oxide) from Na(+)-NQR, which indicates that these residues do not participate directly in the ubiquinone binding site but probably control its accessibility. Indeed, redox-induced difference spectroscopy showed that these mutations prevented the conformational change involved in ubiquinone binding but did not modify the signals corresponding to bound ubiquinone. Moreover, data are presented that demonstrate the NqrA subunit is able to bind ubiquinone but with a low non-catalytically relevant affinity. It is also suggested that Na(+)-NQR contains a single catalytic ubiquinone binding site and a second site that can bind ubiquinone but is not active.
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http://dx.doi.org/10.1074/jbc.M114.574640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4156058PMC
August 2014

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

Science 2012 Nov;338(6111):1209-13

Department of Organic and Biological Chemistry, School of Chemistry, University of Bristol, Bristol BS8 1TS, UK.

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation.
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http://dx.doi.org/10.1126/science.1228633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4658703PMC
November 2012

Protein flexibility directs DNA recognition by the papillomavirus E2 proteins.

Nucleic Acids Res 2011 Apr 3;39(7):2969-80. Epub 2010 Dec 3.

School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK.

Although DNA flexibility is known to play an important role in DNA-protein interactions, the importance of protein flexibility is less well understood. Here, we show that protein dynamics are important in DNA recognition using the well-characterized human papillomavirus (HPV) type 6 E2 protein as a model system. We have compared the DNA binding properties of the HPV 6 E2 DNA binding domain (DBD) and a mutant lacking two C-terminal leucine residues that form part of the hydrophobic core of the protein. Deletion of these residues results in increased specific and non-specific DNA binding and an overall decrease in DNA binding specificity. Using (15)N NMR relaxation and hydrogen/deuterium exchange, we demonstrate that the mutation results in increased flexibility within the hydrophobic core and loop regions that orient the DNA binding helices. Stopped-flow kinetic studies indicate that increased flexibility alters DNA binding by increasing initial interactions with DNA but has little or no effect on the structural rearrangements that follow this step. Taken together these data demonstrate that subtle changes in protein dynamics have a major influence on protein-DNA interactions.
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http://dx.doi.org/10.1093/nar/gkq1217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074142PMC
April 2011
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