Katherine Henzler-Wildman's research while affiliated with University of Wisconsin–Madison and other places
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Publications (57)
Small molecule manipulation of the protein kinase-like (PKL) superfamily has emerged as a remarkably effective therapeutic strategy, with more than 70 new drugs approved in the past 20 years, and has aided mechanistic studies of PKL function. However, such tools are still lacking for many PKL members, including the growing number of atypical kinase...
A critical part of ion channel function is the ability to open and close in response to stimuli, and thus conduct ions in a regulated fashion. While X-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix b...
Small molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize inhibitor and activator molecules for the archety...
The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand H N –F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphoni...
The SARS-CoV-2 RNA genome contains a 5′-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2′-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the kinas...
The SARS-CoV-2 RNA genome contains a 5′-cap that facilitates translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made is not completely understood. Here, we reconstitute the SARS-CoV-2 7MeGpppA2′-O-Me-RNA cap using virally encoded non-structural proteins (nsps). We show that the ki...
Small multidrug resistance (SMR) transporters perform coupled antiport of protons and toxic substrates, contributing to antibiotic resistance through efflux of these compounds from the bacterial cytoplasm. Extensive biophysical studies of the molecular transport mechanism of the E. coli SMR transporter EmrE indicate that it should also be capable o...
The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na ⁺ and K ⁺ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NM...
The solution structure of SARS-CoV-2 nonstructural protein 7 (nsp7) at pH 7.0 has been determined by NMR spectroscopy. nsp7 is conserved in the coronavirinae subfamily and is an essential co-factor of the viral RNA-dependent RNA polymerase for active and processive replication. Similar to the previously deposited structures of SARS-CoV-1 nsp7 at ac...
Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transpor...
The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR...
The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, mak...
Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis...
The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project covid19-nmr, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein nsp7. The 83 amino acid nsp7 protein is an essential cofactor in the RNA-dependent RNA polymerase. The polym...
The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport....
Small multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells. These transporters were recently divided into two subfamilies: the GdX-like and EmrE-like SMRs. The EmrE-like subfamily of SMRs is predicted to contain transporters that are highly promiscuous in both substrate specificity and mechanism based on extensive ch...
Transport stoichiometry provides insight into the mechanism and function of ion-coupled transporters, but measuring transport stoichiometry is time-consuming and technically difficult. With the increasing evidence that many ion-coupled transporters employ multiple transport stoichiometries under different conditions, improved methods to determine t...
Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mecha...
The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. T...
Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alter...
Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoi...
Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detectio...
EmrE is a small multidrug resistance transporter found in Escherichia coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton...
EmrE is a small multidrug resistance transporter found in E. coli that confers resistance to toxic polyaromatic cations due to its proton-coupled antiport of these substrates. Here we show that EmrE breaks the rules generally deemed essential for coupled antiport. NMR spectra reveal that EmrE can simultaneously bind and cotransport proton and drug....
NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that make...
Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular cons...
The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its "active-site" residues-glutamate 14 (Glu14) from each subunit-must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To a...
The NaK channel is small bacterial non-selective cation channel that has proved to be an excellent model system to study basic biochemical and biophysical question concerning ion channels. NaK has been crystalized in the closed state and as an open channel using a truncated construct (NaKΔ19) missing the M0 helix. NaK shares a homologous architectu...
EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active trans- port. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previous...
The small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein i...
EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and
protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized
by this transporter. We have studied a series of tetrahedral compounds, as well as several planar...
Plasmodium parasites use specialized ligands which bind to red blood cell (RBC) receptors during invasion. Defining the mechanism of receptor recognition is essential for the design of interventions against malaria. Here, we present the structural basis for Duffy antigen (DARC) engagement by P. vivax Duffy binding protein (DBP). We used NMR to map...
Despite its apparent simplicity, the potassium channel KcsA possesses a relatively complex gating cycle. Previous studies show conformational changes at the inner gate with an increase in distance from the central axis of symmetry when activated. These changes, coupled to rearrangements at the selectivity filter, are hypothesized to underlie activa...
Reconstitution of integral membrane proteins into membrane mimetic environments suitable for biophysical and structural studies has long been a challenge. Isotropic bicelles promise the best of both worlds-keeping a membrane protein surrounded by a small patch of bilayer-forming lipids while remaining small enough to tumble isotropically and yield...
EmrE, an E. coli small multidrug resistance transporter, exports a broad range of toxic polyaromatic cations, thus imparting resistance to drug compounds of this type. According to the proposed single-site alternating access model of antiport, EmrE converts between inward- and outward-facing structures during the transport cycle, using the import o...
Small multidrug resistance transporters provide an ideal system to study the minimal requirements for active transport. EmrE is one such transporter in Escherichia coli. It exports a broad class of polyaromatic cation substrates, thus conferring resistance to drug compounds matching this chemical description. However, a great deal of controversy ha...
The small multidrug resistance transporters represent a unique model system for studying the mechanism of secondary active transport and membrane protein evolution. However, this seemingly simple protein has been highly controversial. Recent studies have provided experimental evidence that EmrE exists as an asymmetric dimer that exchanges between i...
Bacterial antibiotic resistance is a growing public health concern. One mechanism of resistance arises through drug export by multidrug resistance transporters. To fully understand the function of these proteins requires multiple structures plus kinetic and thermodynamic data to characterize the transport cycle. NMR offers a unique tool to obtain a...
Characterization of the oligomerization of membrane-associated peptides is important to understand the folding and function of biomolecules like antimicrobial peptides, fusion peptides, amyloid peptides, toxins, and ion channels. However, this has been considered to be very difficult, because the amphipathic properties of the constituents of the ce...
LL-37 is the only cathelicidin-derived polypeptide found in humans. Its eclectic function makes this peptide one of the most intriguing chemical defense agents, with crucial roles in moderating inflammation, promoting wound healing, and boosting the human immune system. LL-37 kills both prokaryotic and eukaryotic cells through physical interaction...
The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate-enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical me...
The synergy between structure and dynamics is essential to the function of biological macromolecules. Thermally driven dynamics on different timescales have been experimentally observed or simulated, and a direct link between micro- to milli-second domain motions and enzymatic function has been established. However, very little is understood about...
Because proteins are central to cellular function, researchers have sought to uncover the secrets of how these complex macromolecules execute such a fascinating variety of functions. Although static structures are known for many proteins, the functions of proteins are governed ultimately by their dynamic character (or 'personality'). The dream is t...
Human cathelicidin-derived LL-37 is a 37-residue cationic, amphipathic alpha-helical peptide. It is an active component of mammalian innate immunity. LL-37 has several biological functions including a broad spectrum of antimicrobial activities and LPS-neutralizing activity. In order to determine the high-resolution three-dimensional structure of LL...
A fundamental question is how enzymes can accelerate chemical reactions. Catalysis is not only defined by actual chemical steps, but also by enzyme structure and dynamics. To investigate the role of protein dynamics in enzymatic turnover, we measured residue-specific protein dynamics in hyperthermophilic and mesophilic homologs of adenylate kinase...
LL-37 is a cationic, amphipathic alpha-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium ((2)H) NMR experiments on acyl chain perdeuterated lipids de...
Citations
... In SARS-CoV-2, the Asn1 residue of nsp9, an essential RNA-binding protein, is a target for NiRAN, and substitutions of nsp9 Asn1 and the catalytic residues in AS2 block virus propagation in cell culture [19,34]. NiRAN has also been reported to mediate viral RNA capping, either directly [18] or via an RNA-nsp9 intermediate [22]. Small molecules could interfere with NiRAN activity by binding to nsp9, AS2, or an allosteric site. ...
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... After the 1950s, the dosing, formulation, and administration methods of anti-microbial were expanded. [1,2] Additionally, the mechanism of action of anti-microbial was re-investigated in detail. Nevertheless, meanwhile the start of the 1990s and the 2000s, anti-microbial resistance has increased to importance. ...
... In contrast, for potassium permeation the symmetric pore with all subunits in the crystal conformation is required in NaK (Shi et al., 2018). A recent study characterized the structural plasticity of NaK on multiple timescales using solution NMR (Lewis et al., 2021). The results from this study confirmed the ion dependence of the SF, which was even shown to continue along an allosteric pathway that couples the SF conformation to the lower region of the channel. ...
... N protein N-terminal domain (NTD). Escherichia coli BL21 (DE3) was transformed with a pET28a His-tagged construct (GenScript New Jersey, USA) encoding for the amino acid residues 44 to 180 of the N-terminal RNAbinding globular domain of the Nucleocapsid protein, N (N-NTD) 52 . Bacteria were grown in LB medium with 50 μg/ml kanamycin. ...
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... Second, large conformational changes are not a requisite for the high heat sensitivity of TRPV1. Previous and current studies demonstrate that changes to even a single amino acid may give rise to large effects on heat sensing 14,23,50 . We posit that the exquisitely fine-tuned interaction network renders TRPV1 functionally sensitive to small conformational changes. ...
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... In addition to revealing the binding mode of the guanidinyl headgroup, the structure of Gdx-Clo with octylguanidinium showed that hydrophobic repacking of residues lining one side of the binding pocket opens a portal from the substrate binding site to the membrane interior, accommodating the substrate's long alkyl tail. In addition, a model of an EmrE mutant with reduced conformational exchange dynamics, S64V, computed from extensive NMR measurements, was also reported recently (Shcherbakov et al., 2021). ...
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... Four helices defined by PROCHECK 19 -α1 (V11-L20), α2 (K27-L41), α3 (T45-M62), and α4 (I68-R79) -are aligned with the TALOS-N prediction (green regions) reported in a previous paper. 20 The statistics for the final 20 lowest energy models are shown in Table 1. Only the backbone and heavy atoms of residues in the ordered region (S10-R79) were included in the calculation of RMSDs. ...
... In contrast, a large inward-facing F 4 -TPP + gradient reverses the net transport direction, indicating that protons are driven out of the liposomes against their concentration gradient. This reversal of current is indicative of coupled transport 35,36 and demonstrates that F 4 -TPP + is antiported by EmrE. Although the timescale of transport differs between transporters, a similar reversal of current is observed for proton/guanidinium antiport by the EmrE homolog Gdx 36 . ...
... Biochemical and biophysical data have shown that the transport process of EmrE is highly complex. For example, in addition to acting as a proton-coupled antiporter, EmrE can also function as a proton-coupled symporter or uncoupled uniporter under different conditions [15][16][17][18][19] . Elucidating the mechanism of membrane transport by EmrE requires atomicresolution structural information for multiple states of the protein, as well as dynamics information about the protein and the ligands throughout the transport cycle. ...
... The findings of Zubcevic and Lee [119] and Chen et al. [120], in light of the results discussed in this paper, suggest that rigid voltage sensors (v. gr. ...
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