Debashish Sahu

Asymmetrical roles of zinc fingers in dynamic DNA-scanning process by the inducible transcription factor Egr-1

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals and vascular stresses. Using spectroscopic and computational approaches, we have studied structural, dynamic, and kinetic aspects of the DNA-scanning process in which Egr-1 is nonspecifically bound to DNA and perpetually changes its location on DNA. Our NMR data indicate that Egr-1 undergoes highly dynamic domain motions when scanning DNA. In particular, the zinc finger 1 (ZF1) of Egr-1 in the nonspecific complex is mainly dissociated from DNA and undergoes collective motions on a nanosecond timescale, whereas zinc fingers 2 and 3 (ZF2 and ZF3, respectively) are bound to DNA. This was totally unexpected because the previous crystallographic studies of the specific complex indicated that all of Egr-1’s three zinc fingers are equally involved in binding to a target DNA site. Mutations that are expected to enhance ZF1’s interactions with DNA and with ZF2 were found to reduce ZF1’s domain motions in the nonspecific complex suggesting that these interactions dictate the dynamic behavior of ZF1. By experiment and computation, we have also investigated kinetics of Egr-1’s translocation between two nonspecific DNA duplexes. Our data on the wild type and mutant proteins suggest that the domain dynamics facilitate Egr-1’s intersegment transfer that involves transient bridging of two DNA sites. These results shed light on asymmetrical roles of the zinc finger domains for Egr-1 to scan DNA efficiently in the nucleus.

Bhageerath: an energy based web enabled computer software suite for limiting the search space of tertiary structures of small globular proteins

We describe here an energy based computer software suite for narrowing down the search space of tertiary structures of small globular proteins. The protocol comprises eight different computational modules that form an automated pipeline. It combines physics based potentials with biophysical filters to arrive at 10 plausible candidate structures starting from sequence and secondary structure information. The methodology has been validated here on 50 small globular proteins consisting of 2–3 helices and strands with known tertiary structures. For each of these proteins, a structure within 3–6 Å RMSD (root mean square deviation) of the native has been obtained in the 10 lowest energy structures. The protocol has been web enabled and is accessible at .

Redox properties of the A-domain of the HMGB1 protein

MINT‐6795963: txn (uniprotkb:P10599) and HMGB1 (uniprotkb:P09429) bind (MI:0408) by nuclear magnetic resonance (MI:0077)

Real-time Kinetics of High-mobility Group Box 1 (HMGB1) Oxidation in Extracellular Fluids Studied by in Situ Protein NMR Spectroscopy

Background: Redox of extracellular HMGB1 protein plays an important role in inflammation. Results: The half-life of all-thiol HMGB1 was ∼17 min in serum and saliva and significantly longer in cancer cell culture medium and was modulated by exogenous ligands (e.g. heparin). Conclusion: The extracellular environment dictates HMGB1 oxidation kinetics. Significance: Our approach permits investigating protein oxidation in situ. Some extracellular proteins are initially secreted in reduced forms via a non-canonical pathway bypassing the endoplasmic reticulum and become oxidized in the extracellular space. One such protein is HMGB1 (high-mobility group box 1). Extracellular HMGB1 has different redox states that play distinct roles in inflammation. Using a unique NMR-based approach, we have investigated the kinetics of HMGB1 oxidation and the half-lives of all-thiol and disulfide HMGB1 species in serum, saliva, and cell culture medium. In this approach, salt-free lyophilized 15N-labeled all-thiol HMGB1 was dissolved in actual extracellular fluids, and the oxidation and clearance kinetics were monitored in situ by recording a series of heteronuclear 1H-15N correlation spectra. We found that the half-life depends significantly on the extracellular environment. For example, the half-life of all-thiol HMGB1 ranged from ∼17 min (in human serum and saliva) to 3 h (in prostate cancer cell culture medium). Furthermore, the binding of ligands (glycyrrhizin and heparin) to HMGB1 significantly modulated the oxidation kinetics. Thus, the balance between the roles of all-thiol and disulfide HMGB1 proteins depends significantly on the extracellular environment and can also be artificially modulated by ligands. This is important because extracellular HMGB1 has been suggested as a therapeutic target for inflammatory diseases and cancer. Our work demonstrates that the in situ protein NMR approach is powerful for investigating the behavior of proteins in actual extracellular fluids containing an enormous number of different molecules.

The role of human Dicer-dsRBD in processing small regulatory RNAs.

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.

NMR Studies of Translocation of the Zif268 Protein between Its Target DNA Sites

Zif268 is a zinc-finger protein containing three Cys(2)-His(2)-type zinc-finger domains that bind the target DNA sequence GCGTGGGCG in a cooperative manner. In this work, we characterized translocation of the Zif268 protein between its target DNA sites using NMR spectroscopy. The residual dipolar coupling data and NMR chemical shift data suggested that the structure of the sequence-specific complex between Zif268 and its target DNA in solution is the same as the crystal structure. Using two-dimensional heteronuclear (1)H-(15)N correlation spectra recorded with the fast acquisition method, we analyzed the kinetics of the process in which the Zif268 protein transfers from a target site to another on a different DNA molecule on a minute to hour time scale. By globally fitting the time-course data collected at some different DNA concentrations, we determined the dissociation rate constant for the specific complex and the second-order rate constant for direct transfer of Zif268 from one target site to another. Interestingly, direct transfer of the Zif268 protein between its target sites is >30000-fold slower than corresponding direct transfers of the HoxD9 and the Oct-1 proteins, although the affinities of the three proteins to their target DNA sites are comparable. We also analyzed translocation of the Zif268 protein between two target sites on the same DNA molecules. The populations of the proteins bound to the target sites were found to depend on locations and orientations of the target sites.

Generating NMR chemical shift assignments of intrinsically disordered proteins using carbon-detected NMR methods

There is an extraordinary need to describe the structures of intrinsically disordered proteins (IDPs) due to their role in various biological processes involved in signaling and transcription. However, general study of IDPs by NMR spectroscopy is limited by the poor (1)H amide chemical shift dispersion typically observed in their spectra. Recently, (13)C direct-detected NMR spectroscopy has been recognized as enabling broad structural study of IDPs. Most notably, multidimensional experiments based on the (15)N,(13)C CON spectrum make complete chemical shift assignment feasible. Here we document a collection of NMR-based tools that efficiently lead to chemical shift assignment of IDPs, motivated by a case study of the C-terminal disordered region from the human pancreatic transcription factor Pdx1. Our strategy builds on the combination of two three-dimensional (3D) experiments, (HN-flip)N(CA)CON and 3D (HN-flip)N(CA)NCO, that enable daisy chain connections to be built along the IDP backbone, facilitated by acquisition of amino acid-specific (15)N,(13)C CON-detected experiments. Assignments are completed through carbon-detected, total correlation spectroscopy (TOCSY)-based side chain chemical shift measurement. Conducting our study required producing valuable modifications to many previously published pulse sequences, motivating us to announce the creation of a database of our pulse programs, which we make freely available through our website.

Ensemble analysis of primary microRNA structure reveals an extensive capacity to deform near the Drosha cleavage site.

Most noncoding RNAs function properly only when folded into complex three-dimensional (3D) structures, but the experimental determination of these structures remains challenging. Understanding of primary microRNA (miRNA) maturation is currently limited by a lack of determined structures for nonprocessed forms of the RNA. SHAPE chemistry efficiently determines RNA secondary structural information with single-nucleotide resolution, providing constraints suitable for input into MC-Pipeline for refinement of 3D structure models. Here we combine these approaches to analyze three structurally diverse primary microRNAs, revealing deviations from canonical double-stranded RNA structure in the stem adjacent to the Drosha cut site for all three. The necessity of these deformable sites for efficient processing is demonstrated through Drosha processing assays. The structure models generated herein support the hypothesis that deformable sequences spaced roughly once per turn of A-form helix, created by noncanonical structure elements, combine with the necessary single-stranded RNA-double-stranded RNA junction to define the correct Drosha cleavage site.

Observing In-phase Single-Quantum 15N Multiplets for NH2/NH3+ Groups with Two-dimensional Heteronuclear Correlation Spectroscopy

Two-dimensional (2D) F1-(1)H-coupled HSQC experiments provide 3:1:1:3 and 1:0:1 multiplets for AX(3) and AX(2) spin systems, respectively. These multiplets occur because, in addition to the 2S(y)H(z)(a)-->2S(y)H(z)(a) process, the coherence transfers such as 2S(y)H(z)(a)-->2S(y)H(z)(b) occurring in t(1) period provide detectable magnetization during the t(2) period. Here, we present a 2D F1-(1)H-coupled (1)H-(15)N heteronuclear correlation experiment that provides a 1:3:3:1 quartet for AX(3) spin system and a 1:2:1 triplet for AX(2). The experiment is a derivative of 2D HISQC experiment [J. Iwahara, Y.S. Jung, G.M. Clore, Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation. J. Am. Chem. Soc. 129 (2007) 2971-2980] and contains a scheme that kills anti-phase single-quantum terms generated in the t(1) period. The purge scheme is essential to observe in-phase single-quantum multiplets. Applications to the NH(2) and NH(3)(+) groups in proteins are demonstrated.

Assessing Coupled Protein Folding and Binding Through Temperature-Dependent Isothermal Titration Calorimetry

Broad interest in the thermodynamic driving forces of coupled macromolecular folding and binding is motivated by the prevalence of disorder-to-order transitions observed when intrinsically disordered proteins (IDPs) bind to their partners. Isothermal titration calorimetry (ITC) is one of the few methods available for completely evaluating the thermodynamic parameters describing a protein-ligand binding event. Significantly, when the effective ΔH° for the coupled folding and binding process is determined by ITC in a temperature series, the constant-pressure heat capacity change (ΔCp) associated with these coupled equilibria is experimentally accessible, offering a unique opportunity to investigate the driving forces behind them. Notably, each of these molecular-scale events is often accompanied by strongly temperature-dependent enthalpy changes, even over the narrow temperature range experimentally accessible for biomolecules, making single temperature determinations of ΔH° less informative than typically assumed. Here, we will document the procedures we have adopted in our laboratory for designing, executing, and globally analyzing temperature-dependent ITC studies of coupled folding and binding in IDP interactions. As a biologically significant example, our recent evaluation of temperature-dependent interactions between the disordered tail of FCP1 and the winged-helix domain from Rap74 will be presented. Emphasis will be placed on the use of publically available analysis programs written in MATLAB that facilitate quantification of the thermodynamic forces governing IDP interactions. Although motivated from the perspective of IDPs, the experimental design principles and data fitting procedures presented here are general to the study of most noncooperative ligand binding equilibria.