Research Spotlight

Yuan He Lab

Structural Mechanism of ATP-independent Transcription Initiation by RNA Polymerase I

Yan Han, Chunli Yan, Thi Hoang Duong Nguyen, Ashleigh J. Jackobel, Ivaylo Ivanov, Bruce Knutson, Yuan He

Transcription initiation by RNA Polymerase I (Pol I) depends on the Core Factor (CF) complex to recognize the upstream promoter and assemble into a Pre-Initiation Complex (PIC). Here, we solve a structure of Saccharomyces cerevisiae Pol I-CF-DNA to 3.8Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals a bipartite architecture of Core Factor and its recognition of the promoter from -27 to -16. Core Factor’s intrinsic mobility correlates well with different conformational states of the Pol I cleft, in addition to the stabilization of either the Rrn7 N-terminal domain near Pol I wall or the tandem winged helix domain of A49 at a partially overlapping location. Comparison of the three states in this study with the Pol II system suggests that a ratchet motion of the Core Factor-DNA sub-complex upstream facilitates promoter melting in an ATP-independent manner, distinct from a DNA translocase actively threading the downstream DNA in the Pol II PIC.

Leonard Lab

Contributions of Unique Intracellular Domains to Switchlike Biosensing by Toll-like Receptor 4
February, 2015

Nichole M. Daringer, Kelly A. Schwarz and Joshua N. Leonard

Toll-like receptors (TLRs) mediate immune recognition of both microbial infections and tissue damage. Aberrant TLR signaling promotes disease; thus, understanding the regulation of TLR signaling is of medical relevance. Although downstream mediators of TLR signaling have been identified, the detailed mechanism by which ligand binding-mediated dimerization induces downstream signaling remains poorly understood. Here, we investigate this question for TLR4, which mediates responsiveness to bacterial LPS and drives inflammatory disease. TLR4 exhibits structural and functional features that are unique among TLRs, including responsiveness to a wide variety of ligands. However, the connection between these structural features and the regulation of signaling is not clear. Here, we investigated how the unique intracellular structures of TLR4 contribute to receptor signaling. Key conclusions include the following. 1) The unique intracellular linker of TLR4 is important for achieving LPS-inducible signaling via Toll/IL-1 receptor (TIR) domain-containing adapter-inducing interferon-β (TRIF) but less so for signaling via myeloid differentiation primary response 88 (MyD88). 2) Membrane-bound TLR4 TIR domains were sufficient to induce signaling. However, introducing long, flexible intracellular linkers neither induced constitutive signaling nor ablated LPS-inducible signaling. Thus, the initiation of TLR4 signaling is regulated by a mechanism that does not require tight geometric constraints. Together, these observations necessitate refining the model of TLR4 signal initiation. We hypothesize that TLR4 may interact with an inhibitory partner in the absence of ligand, via both TIR and extracellular domains of TLR4. In this speculative model, ligand binding induces dissociation of the inhibitory partner, triggering spontaneous, switchlike TIR domain homodimerization to initiate downstream signaling.



Rice Group

Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro
September, 2013

Julian L. Klosowiak, Pamela J. Focia, Srinivas Chakravarthy, Eric C. Landahl, Douglas M. Freymann & Sarah E. Rice

The outer mitochondrial membrane protein Miro is a highly conserved calcium-binding GTPase that is at the regulatory nexus of several processes, including mitochondrial transport and autophagy. Miro attaches mitochondria to the microtubule-based motor protein kinesin-1 and acts as a calcium-dependent switch for mitochondrial movement. Phosphorylation of Miro by Pink1 kinase and its subsequent Parkin-mediated degradation leads to mitophagy of damaged mitochondria. Relatively little is known about the molecular underpinnings of these processes and a structural understanding of the relevant protein machinery is lacking. Here we present crystal structures comprising the tandem EF hand and C-terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified “hidden” EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand/cGTPase interface and may have implications for Pink1-mediated recruitment of Parkin to the mitochondrial surface. Our results suggest structural mechanisms for calcium, nucleotide, and phosphorylation-dependent regulation of mitochondrial function by Miro.

EMBO reports, 27 September 2013, doi:10.1038/embor.2013.151

Kelleher Group

Total Kinetic Analysis Reveals How Combinatorial Methylation Patterns are Established on Lysines 27 and 36 of Histone H3
July, 2012

The great DNA sequencing technology leap in recent years has made it possible to sequence more and more genome from individual cancer patients. The rationale behind these great efforts is that the recurring mutations, constantly showing up in many cancer patients, are the "driver" mutations likely to cause cancer whereas rare mutations are just "passenger" mutations and therefore not critical. Surprisingly, mutations in genes encoding histone modifying enzymes turn out to be such "driver" mutations. These enzymes can "write" and "erase" some small tags (methylation, acetylation, phosphorylation, etc.) in the tail of histones, which pack our DNA into the form of chromatin. The complex combinations of these different modifications and many different outcomes dictated by various "readers" have been the central part of "histone code" theory.

Importantly, research suggests that abnormal chromatin landscape caused by these recurring mutations is the molecular culprit leading to cancer since histone modification can influence how cells use the information stored in DNA. Because this process is not involved in the sequence of DNA itself, it is referred as epigenetic. In contrast to the genetic mutations, histone modifications are reversible. Therefore, we can restore the chromatin landscape from disease state to normal state if we can manipulate the "writing" and "erasing" speed of deregulated histone modifying enzymes.

To study how aberrant histone modifications are established in multiple myeloma patients with overproduction of histone methyltransferase (MMSET) as result of chromosome translocation, we developed an integrated approach Mass Spectrometry-based Measurement and Modeling of Histone Methylation Kinetics ("M4K") by combining stable isotopes, quantitative mass spectrometry and computational modeling to determine the full matrix of effective rate constants for two methylation sites at K27 and K36 of histone H3. M4K revealed that when H3K36 or H3K27 is dimethylated, then rates of further methylation on the other site are reduced precipitously, which explains why both H3K27 and H3K36 methylation patterns are affected in multiple myeloma (bi-directional antagonism model). Another surprise discovery is the increase methylation turnover at H3K27 when MMSET is overexpressed. Most significantly, we can use the kinetic model obtained by M4K to predict how much inhibition is required to restore the normal epigenetic state in these cancer cells.


Rosenzweig Lab

Structural Basis for Activation of Class Ib Ribonucleotide Reductase
September, 2010

Amie K. Boal, Joseph A. Cotruvo, Jr., JoAnne Stubbe, and Amy C. Rosenzweig

The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a MnIII2-tyrosyl radical (Y•) or a FeIII2-Y• cofactor in the NrdF subunit.  Whereas FeIII2-Y• can self-assembleStructural basis for activation of class Ib ribonucleotide reductase from FeII2-NrdF and O2, activation of MnII2-NrdF requires a reducedflavoprotein, NrdI,proposed to form the oxidant for cofactor assembly by reduction of O2.  The crystal structures reported here of E. coli MnII2-NrdF and FeII2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation.  In the structures of MnII2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF MnII2 active site.  Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of MnIII2-Y• cofactor assembly.

Science, in press, Sciencexpress, August 5, 2010 (PDF)


Hoffman Lab

Photoinitiated Singlet and Triplet Electron Transfer Across a Re-Designed [Myoglobin, Cytochrome b5] Interface
February, 2010

Judith M. Nocek, Amanda K. Knutson, Peng Xiong, Nadia Petlakh Co, and Brian M. Hoffman

Abstract: We describe a strategy by which reactive binding of a weakly-bound, ‘dynamically docked (DD)’ complex without a known structure can be strengthened electrostatically through optimized placement of surface charges, and discuss its use in modulating complex formation between Photoinitiated Singlet and Triplet Electron Transfer Across a Re-Designed [Myoglobin, Cytochrome b5] Interfacemyoglobin (Mb) and cytochrome b5 (b5). The strategy employs paired Brownian Dynamics (BD) simulations, one which monitors overall binding, the other reactive binding, to examine [X g K] mutations on the surface of the partners, with a focus on single and multiple [D/E g K] charge reversal mutations. This procedure has been applied to the [Mb, b5] complex, indicating mutations of Mb residues D44, D60 and E85 to be the most promising, with combinations of these showing a nonlinear enhancement of reactive binding. A novel method of displaying BD profiles shows that the ‘hits’ of b5 on the surfaces of Mb(WT), Mb(D44K/D60K), and Mb(D44K/D60K/E85K) progressively coalesce into two ‘clusters’: a ‘diffuse’ cluster of hits that are distributed over the Mb surface and have negligible electrostatic binding energy; a ‘reactive’ cluster of hits with considerable stability that are localized near its heme edge, with short Fe-Fe distances favorable to electron transfer (ET). Thus binding and reactivity progressively become correlated by the mutations. This finding fits well with recent proposals that complex formation is a two-step process, proceeding through the formation of a weakly-bound encounter complex (‘diffuse cluster’) to a well-defined bound complex (‘reactive cluster’). The design procedure has been tested through measurements of photoinitiated ET between the Zn-substituted forms of Mb(WT), Mb(D44K/D60K) and Mb(D44K/D60K/E85K) and Fe3+b5. Both mutants convert the complex from the DD regime exhibited by Mb(WT), in which the transient complex is in fast kinetic exchange with its partners, koff >> ket, to the slow-exchange regime, ket >> koff, and both mutants exhibit rapid intracomplex ET from the triplet excited state to Fe3+b5 (rate constant, ket ~ 106 s-1). The affinity constants of the mutant Mbs cannot be derived through conventional analysis procedures because intracomplex singlet ET quenching causes the triplet-ground absorbance difference to progressively decrease during a titration, but this effect has been incorporated into a new procedure for computing binding constants. Most importantly, this is the first evidence for photo-induced singlet ET across a protein-protein interface.


Widom Lab

From DNA sequence to transcriptional behaviour: a quantitative approach
June, 2009

Eran Segal and Jonathan Widom

Abstract: Complex transcriptional behaviours are encoded in the DNA sequences of gene regulatory regions. Advances in our understanding of these behaviours have been recently gained through quantitative models that From DNA sequence to transcriptional behaviout: a quantitative approachdescribe how molecules such as transcription factors and nucleosomes interact with genomic sequences. An emerging view is that every regulatory sequence is associated with a unique binding affinity landscape for each molecule and, consequently, with a unique set of molecule-binding configurations and transcriptional outputs. We present a quantitative framework based on existing methods that unifies these ideas. This framework explains many experimental observations regarding the binding patterns of factors and nucleosomes and the dynamics of transcriptional activation. It can also be used to model more complex phenomena such as transcriptional noise and the evolution of transcriptional regulation.


Hoffman Lab

Electrostatic Redesign of the [Myoglobin,Cytochrome b5] Interface To Create a Well-Defined Docked Complex with Rapid Interprotein Electron Transfer
June, 2009 Electrostatic Redesign of the [Myoglobin,Cytochrome b5] Interface To Create a Well-Defined Docked Complex with Rapid Interprotein Electron Transfer

Peng Xiong, Judith M. Nocek, Amanda K. K. Griffin, Jingyun Wang, and Brian M. Hoffman

Full text here


Matouschek Lab

Substrate selection by the proteasome during degradation of protein complexes
January, 2008

The proteasome controls the turnover of many cellular proteins. Two structural features are typically required for proteins to be degraded: covalently attached ubiquitin polypeptides that allow binding to the proteasome and an unstructured region in the targeted protein that initiates proteolysis. Matouschek LabHere, we have tested the degradation of model proteins to further explore how the proteasome selects its substrates. Using purified yeast proteasome and mammalian proteasome in cell lysate, we have demonstrated that the two structural features can act in trans when separated onto different proteins in a multisubunit complex. In such complexes, the location of the unstructured initiation site and its chemical properties determine which subunit is degraded. Thus, our findings reveal the molecular basis of subunit specificity in the degradation of protein complexes. In addition, our data provide a plausible explanation for how adaptor proteins can bind to otherwise stable proteins and target them for degradation.

Marko and Mondragon Groups

November, 2007

Topoisomerases are enzymes that remove topological constraints in double-stranded DNA introduced by processes such as replication, recombination topoisomerases and transcription. The mechanism of action of Topoisomerase V, a new topoisomerase unrelated to other topoisomerases, has remained unknown. The Mondragon and Marko groups now show via single-molecule experiments that Topoisomerase V employs a similar mechanism as type IB topoisomerases, but using a completely different structural framework.


Rice Lab

The kinesin-1 motor protein is regulated by a direct interaction of its head and tail
July, 2008

The motor protein kinesin-1 has a regulatory tail domain that can simultaneously contact the enzymatically critical kinesin-1 motor proteinSwitch I region of its motile head domains and the microtubule, according to an 8 Å cryo-EM reconstruction obtained by Dietrich et al. (Rice lab).  These interactions suggest a mechanism for tail-mediated regulation of kinesin-1's ATPase activity and raise the possibility of a paused state of kinesin-1 on microtubules.


Areas of Research

Many of the research programs of Northwestern's Biophysics Faculty fall within the realms of Structural Biology, Drug Design, Quantitative and Mechanistic Biology, Computational Biology and Chemical Biology. With over 20 faculty involved in the program and mani inter-laboratory collaborations, a variety of fundamental biological questions are being studied, including:

  • Nucleic acid structure and function
  • Mechanisms of gene regulation
  • Protein and RNA processing in the cell
  • Intracellular metal trafficking
  • Molecular mechanisms of viral infection
  • Mechanisms of macromolecular machines
  • Membrane protein structure and function

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