The structural biology and dynamics of malate dehydrogenases.

Malate dehydrogenase (MDH) enzymes catalyze the reversible oxidoreduction of malate to oxaloacetate using NAD(P) as a cofactor. This reaction is vital for metabolism and the exchange of reducing equivalents between cellular compartments. There are more than 100 structures of MDH in the Protein Data Bank, representing species from archaea, bacteria, and eukaryotes. This conserved family of enzymes shares a common nucleotide-binding domain, substrate-binding domain, and subunits associate to form a dimeric or a tetrameric enzyme. Despite the variety of crystallization conditions and ligands in the experimental structures, the conformation and configuration of MDH are similar. The quaternary structure and active site dynamics account for most conformational differences in the experimental MDH structures. Oligomerization appears essential for activity despite each subunit having a structurally independent active site. There are two dynamic regions within the active site that influence substrate binding and possibly catalysis, with one of these regions adjoining the subunit interface. In this review, we introduce the reader to the general structural framework of MDH highlighting the conservation of certain features and pointing out unique differences that regulate MDH enzyme activity.

Length of course-based undergraduate research experiences (CURE) impacts student learning and attitudinal outcomes: A study of the Malate dehydrogenase CUREs Community (MCC).

Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students.

External Collaboration Results in Student Learning Gains and Positive STEM Attitudes in CUREs.

The implementation of course-based undergraduate research experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasingly collaborative nature of research. While some CUREs have expanded, involving multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no-CURE, 2) single-institution CURE (CURE), and 3) external collaborative CURE (ec-CURE), and assessed academic and attitudinal outcomes. The ec-CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypotheses and discuss their data. The sharing of ideas, data, and materials with an external faculty member allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec-CURE had the greatest gains in experimental design; self-reported course benefits; scientific skills; and science, technology, engineering, and mathematics (STEM) importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2- and 4-year institutions, illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.

Predicting and validating a model of suppressor of IKKepsilon through biophysical characterization.

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3-TANK-binding kinase-1-mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α-actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function. Circular dichroism revealed a primarily (73%) helical structure of minimal stability ( =32°C) but reversibly denatured. Limited proteolysis (LP) and chemical modification identified the N-terminal 2/3 of the protein as dynamic and accessible, whereas size exclusion chromatography (SEC) confirmed three homo-oligomeric species. SEC coupled to chemical crosslinking characterized the primary species as dimeric, a secondary hexameric species, and a higher order aggregate/polymer. Fluorescence polarization using intrinsic tryptophan fluorescence contextualized the anisotropy value for the SIKE dimer (molecular weight 51.8 kDa) among proteins of known structure, bovine serum albumin (BSA; 66 kDa), and glutamate dehydrogenase (GDH; 332 kDa). Radii of gyration for BSA and GDH provided exclusionary values for SIKE tertiary and dimeric quaternary models that otherwise conformed to secondary structure, LP, and modification data. Dimeric quaternary models were further culled using acrylamide quenching data of SIKE's single tryptophan that showed a single, protected environment. The low cooperativity of folding and regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein-binding partners.

Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α-actinin, linking innate immunity to the cytoskeleton.

Suppressor of IKKepsilon (SIKE) is associated with the type I interferon response of the innate immune system through TANK-binding kinase 1 (TBK1). Originally characterized as an endogenous inhibitor of TBK1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that SIKE acts as a high-affinity substrate of TBK1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and SIKE CRISPR/Cas9 knockout HAP1 cells showed an ~ 20% decrease in cell migration. Investigation of the SIKE interaction network through affinity purification/mass spectrometry showed that SIKE formed interactions with cytoskeletal proteins. In immunofluorescence assays, endogenous SIKE localized to cytosolic puncta in both epithelial and myeloid cells and to nuclear puncta in myeloid cells, while in epithelial cells additional staining occurred in stress fiber-like structures and adjacent to the plasma membrane. Using cellular markers, co-occurrence of SIKE fluorescence with actin, α-actinin, and ezrin was detected. Reciprocal immunoprecipitation revealed a SIKE:tubulin interaction sensitive to the phosphorylation state of SIKE, but a SIKE:α-actinin interaction was unchanged by SIKE phosphorylation. In vitro precipitation assays confirmed a direct SIKE interaction with tubulin and α-actinin. These results indicate that SIKE may promote cell migration by directly associating with the cytoskeleton. In this role, SIKE may mediate cytoskeletal rearrangement necessary in innate immunity, but also link a key catalytic hub, TBK1, to the cytoskeleton. DATABASE: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD007262.

CUREs in biochemistry-where we are and where we should go.

Integration of research experience into classroom is an important and vital experience for all undergraduates. These course-based undergraduate research experiences (CUREs) have grown from independent instructor lead projects to large consortium driven experiences. The impact and importance of CUREs on students at all levels in biochemistry was the focus of a National Science Foundation funded think tank. The state of biochemistry CUREs and suggestions for moving biochemistry forward as well as a practical guide (supplementary material) are reported here. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):7-12, 2017.

Cyclic-di-GMP binding induces structural rearrangements in the PlzA and PlzC proteins of the Lyme disease and relapsing fever spirochetes: a possible switch mechanism for c-di-GMP-mediated effector functions.

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R145 of the PilZ domain motif, R145xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions.

Mechanism of endogenous regulation of the type I interferon response by suppressor of IκB kinase epsilon (SIKE), a novel substrate of TANK-binding kinase 1 (TBK1).

TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKε (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (K(i, app) = 350 nM) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar Km value at low substrate concentrations (∼50 nM) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 µM; SIKE = 0.4 µM). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels.

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K (+) currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K (+) currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure.

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.

Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics.

Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR-3, CAOV-3; patient samples malignant 1 and 2) and resistant (DOV-13, SKOV-3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70-fold with malignant 2, 43-fold with OVCAR-3). Using agonists, antagonists, and shRNA-mediated knockdown of dsRNA receptors, we show that TLR3, RIG-I, and mda5 coordinated a caspase 8/9- and interferon-dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5- to 7.5-fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4-0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI=1.5-2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA-sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.

Structure of factor H-binding protein B (FhbB) of the periopathogen, Treponema denticola: insights into progression of periodontal disease.

Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A K(D) value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.

Crystallization of the factor H-binding protein, FhbB, from the periopathogen Treponema denticola.

Treponema denticola is a primary etiological agent of periodontal disease. T. denticola evades complement-mediated killing by binding to the host's factor H (FH), a negative regulator of the alternative complement pathway. The T. denticola FH-binding protein has been identified and designated as factor H-binding protein B (FhbB). Crystals of recombinant FhbB were obtained by the hanging-drop vapor-diffusion method using sodium citrate and 0.2 M sodium thiocyanate. FhbB crystals diffracted to 1.8 Šresolution and belonged to space group P4(3)2(1)2 or P4(1)2(1)2, with unit-cell parameters a = b = 46.76, c = 167.68 Å. Two FhbB molecules per asymmetric unit gave a Matthews coefficient of 2.2 Å(3) Da(-1) and a solvent content of 44%. FhbB is the smallest bacterially produced FH-binding protein identified to date. Determination of its structure will provide unique insight into the minimal structural determinants required for FH binding.

Measuring the effect of ligand binding on the interface stability of multimeric proteins using dynamic light scattering.

We have demonstrated that an approach using guanidine hydrochloride at low concentrations to progressively disrupt protein-protein interactions can be quantitated using dynamic light scattering. This approach is sensitive enough to detect ligand-induced changes of subunit-subunit interactions for homo-hexameric glutamate dehydrogenase, allowing ΔΔG of reversible subunit dissociation to be calculated. The use of dynamic light scattering makes this approach generally applicable to soluble proteins to monitor the relative strength of protein-protein interactions with a particular emphasis on assessing the impact of ligand binding on such interfaces.

Cisplatin enhances protein kinase R-like endoplasmic reticulum kinase- and CD95-dependent melanoma differentiation-associated gene-7/interleukin-24-induced killing in ovarian carcinoma cells.

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a unique interleukin (IL)-10 family cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which recombinant adenoviral delivery of MDA-7/IL-24 inhibits cell survival of human ovarian carcinoma cells. Expression of MDA-7/IL-24 induced phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor2alpha (eIF2alpha). In a PERK-dependent fashion, MDA-7/IL-24 reduced ERK1/2 and AKT phosphorylation and activated c-Jun NH(2)-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK). MDA-7/IL-24 reduced MCL-1 and BCL-XL and increased BAX levels via PERK signaling; cell-killing was mediated via the intrinsic pathway, and cell killing was primarily necrotic as judged using Annexin V/propidium iodide staining. Inhibition of p38 MAPK and JNK1/2 abolished MDA-7/IL-24 toxicity and blocked BAX and BAK activation, whereas activation of mitogen-activated extracellular-regulated kinase (MEK) 1/2 or AKT suppressed enhanced killing and JNK1/2 activation. MEK1/2 signaling increased expression of the MDA-7/IL-24 and PERK chaperone BiP/78-kDa glucose regulated protein (GRP78), and overexpression of BiP/GRP78 suppressed MDA-7/IL-24 toxicity. MDA-7/IL-24-induced LC3-green fluorescent protein vesicularization and processing of LC3; and knockdown of ATG5 suppressed MDA-7/IL-24-mediated toxicity. MDA-7/IL-24 and cisplatin interacted in a greater than additive fashion to kill tumor cells that was dependent on a further elevation of JNK1/2 activity and recruitment of the extrinsic CD95 pathway. MDA-7/IL-24 toxicity was enhanced in a weak additive fashion by paclitaxel; paclitaxel enhanced MDA-7/IL-24 + cisplatin lethality in a greater than additive fashion via BAX. Collectively, our data demonstrate that MDA-7/IL-24 induces an endoplasmic reticulum stress response that activates multiple proapoptotic pathways, culminating in decreased ovarian tumor cell survival.

Predicting Toll-like receptor structures and characterizing ligand binding.

Toll-like receptor (TLR) ligand-binding domains comprise 18-25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.

The TLR3 signaling complex forms by cooperative receptor dimerization.

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40-50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

The dsRNA binding site of human Toll-like receptor 3.

Pathogen recognition by Toll-like receptors (TLRs) initiates innate immune responses that are essential for inhibiting pathogen dissemination and for the development of acquired immunity. The TLRs recognize pathogens with their N-terminal ectodomains (ECD), but the molecular basis for this recognition is not known. Recently we reported the x-ray structure for unliganded TLR3-ECD; however, it has proven difficult to obtain a crystal structure of TLR3 with its ligand, dsRNA. We have now located the TLR3 ligand binding site by mutational analysis. More than 50 single-residue mutations have been generated throughout the TLR3-ECD, but only two, H539E and N541A, resulted in the loss of TLR3 activation and ligand binding functions. These mutations locate the dsRNA binding site on the glycan-free, lateral surface of TLR3 toward the C terminus and suggest a model for dsRNA binding and TLR3 activation.