Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies

In 2015, monoclonal antibodies (mAbs) that selectively recognize the 1-pHis or 3-pHis isoforms of phosphohistidine were developed by immunizing rabbits with degenerate Ala/Gly peptides containing the nonhydrolyzable phosphohistidine (pHis) analog- phosphotriazolylalanine (pTza). Here, we report structures of five rabbit mAbs bound to cognate pTza peptides: SC1-1 and SC50-3 that recognize 1-pHis, and their 3-pHis–specific counterparts, SC39-4, SC44-8, and SC56-2. These cocrystal structures provide insights into the binding modes of the pTza phosphate group that are distinct for the 1- and 3-pHis mAbs with the selectivity arising from specific contacts with the phosphate group and triazolyl ring. The mode of phosphate recognition in the 3-pHis mAbs recapitulates the Walker A motif, as present in kinases. The complementarity-determining regions (CDRs) of four of the Fabs interact with the peptide backbone rather than peptide side chains, thus conferring sequence independence, whereas SC44-8 shows a proclivity for binding a GpHAGA motif mediated by a sterically complementary CDRL3 loop. Specific hydrogen bonding with the triazolyl ring precludes recognition of pTyr and other phosphoamino acids by these mAbs. Kinetic binding experiments reveal that the affinity of pHis mAbs for pHis and pTza peptides is submicromolar. Bound pHis mAbs also shield the pHis peptides from rapid dephosphorylation. The epitope–paratope interactions illustrate how these anti-pHis antibodies are useful for a wide range of research techniques and this structural information can be utilized to improve the specificity and affinity of these antibodies toward a variety of pHis substrates to understand the role of histidine phosphorylation in healthy and diseased states.

Download Full-text

DISCOVERY AND EVALUATION OF POTENTIAL SONIC HEDGEHOG SIGNALING PATHWAY INHIBITORS USING PHARMACOPHORE MODELING AND MOLECULAR DYNAMICS SIMULATIONS

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720011005732 ◽

2011 ◽

Vol 09 (supp01) ◽

pp. 15-35 ◽

Cited By ~ 9

Author(s):

SWAN HWANG ◽

SUNDARAPANDIAN THANGAPANDIAN ◽

YUNO LEE ◽

SUGUNADEVI SAKKIAH ◽

SHALINI JOHN ◽

...

Keyword(s):

Molecular Dynamics ◽

Signaling Pathway ◽

Sonic Hedgehog ◽

Active Site ◽

Hedgehog Signaling ◽

Structural Information ◽

Pharmacophore Modeling ◽

Binding Modes ◽

Shh Signaling ◽

Wide Range

Sonic hedgehog (Shh) plays an important role in the activation of Shh signaling pathway that regulates preservation and rebirth of adult tissues. An abnormal activation of this pathway has been identified in hyperplasia and various tumorigenesis. Hence the inhibition of this pathway using a Shh inhibitor might be an efficient way to treat a wide range of malignancies. This study was done in order to develop a lead chemical candidate that has an inhibitory function in the Shh signaling pathway. We have generated common feature pharmacophore models using three-dimensional (3D) structural information of robotnikinin, an inhibitor of the Shh signaling pathway, and its analogs. These models have been validated with fit values of robotnikinin and its analogs, and the best model was used as a 3D structural query to screen chemical databases. The hit compounds resulted from the screening docked into a proposed binding site of the Shh named pseudo-active site. Molecular dynamics (MD) simulations were performed to investigate detailed binding modes and molecular interactions between the hit compounds and functional residues of the pseudo-active site. The results of the MD simulation analyses revealed that the hit compounds can bind the pseudo-active site with high affinity than robotnikinin. As a result of this study, a candidate inhibitor (GK 03795) was selected as a potential lead to be employed in future Shh inhibitor design.

Download Full-text

Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1715016115 ◽

2018 ◽

Vol 115 (12) ◽

pp. E2706-E2715 ◽

Cited By ~ 12

Author(s):

Saannya Sequeira ◽

Devon Kavanaugh ◽

Donald A. MacKenzie ◽

Tanja Šuligoj ◽

Samuel Walpole ◽

...

Keyword(s):

Lactobacillus Reuteri ◽

Structural Information ◽

Bacterial Species ◽

Surface Proteins ◽

Structural Basis ◽

Gene Encoding ◽

Rhamnogalacturonan I ◽

Host Microbe Interactions ◽

Dynamics Simulations

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates, displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens, but no structural information is available in commensal bacteria. Here we report the 2.00-Å and 1.92-Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique β-solenoid fold in this important adhesin family. SRRP53608-BR bound to host epithelial cells and DNA at neutral pH and recognized polygalacturonic acid (PGA), rhamnogalacturonan I, or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of SRRP53608-BR with PGA. Long molecular dynamics simulations showed that SRRP53608-BR undergoes a pH-dependent conformational change. Together, these findings provide mechanistic insights into the role of SRRPs in host–microbe interactions and open avenues of research into the use of biofilm-forming probiotics against clinically important pathogens.

Download Full-text

Role of interleukin-15 and interleukin-18 in the secretion of sIL-6R and sgp130 by human neutrophils

Mediators of Inflammation ◽

10.1080/0962935031000134905 ◽

2003 ◽

Vol 12 (3) ◽

pp. 179-183 ◽

Cited By ~ 4

Author(s):

E. Jablonska ◽

M. Marcinczyk

Keyword(s):

Monoclonal Antibodies ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Neutrophils ◽

Enzyme Linked Immunosorbent Assay ◽

Cytoplasmic Proteins ◽

Wide Range ◽

Interleukin 15 ◽

Culture Supernatants

Background:Available data indicate that neutrophils (PMN) produce a wide range of cytokines with the potential to modulate immune response. Recent investigation have shown that interleukin (IL)-15 and IL-18 potentiated several functions of normal neutrophils. It has been reported that IL-18-induced cytokine production may be significantly enhanced by coincident addition of IL-15.Aims:In the present study we compared the effect of recombinant human (rh)IL-15 and rhIL-18 as well as effect of a rhIL-15 and rhIL-18 combination on the induction secretion of sIL-6Rα and sgp130 by human neutrophils. Methods: PMN were isolated from heparinized whole blood of healthy persons. The PMN were cultured for 18 h at 37°C in a humidified incubator with 5% CO2. rhIL-15 and/or rhIL-18 and lipopolysaccharide were tested to PMN stimulation. The culture supernatants of PMN were removed and examined for the presence of sIL-6R and sgp130 by human enzyme-linked immunosorbent assay kits. Cytoplasmic protein fractions of PMN were analysed for the presence of sIL-6R and sgp130 by western blotting using monoclonal antibodies capable of detecting these proteins. Cells were lysed and cytoplasmic proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto nitrocellulose and incubated with the primary monoclonal antibodies anti-sIL-6R and anti-sgp130. The membranes were incubated at room temperature with alkaline phosphatase anti-mouse immunoglobulin G. Immunoreactive protein bans were visualized by an AP Conjugate Substrate Kit.Results and conclusion:The results of our investigation revealed that IL-15 alone, similarly to IL-18, has no significant ability for the regulation of both soluble IL-6 receptors, sIL-6R and sgp130, released by human neutrophils. It is interesting to note that the secretion of sgp130 was changed after PMN stimulation with rhIL-15 in the presence of rhIL-18. The combination of rhIL-15 and rhIL-18 was shown to induce PMN to secretion relatively higher amounts of sgp130 compared with the stimulation of PMN with rhIL-15 alone and rhIL-18 alone. The results obtained suggest that IL-15 and IL-18, belonging to the inflammatory cytokines, through the regulation of sgp130 secretion must be also considered as anti-inflammatory mediators that may influence the balance reactions mediated by the IL-6 cytokine family.

Download Full-text

Determinants shaping the nanoscale architecture of the mouse rod outer segment

10.1101/2021.08.18.456753 ◽

2021 ◽

Author(s):

Matthias Pöge ◽

Julia Mahamid ◽

Sanae S Imanishi ◽

Jürgen M Plitzko ◽

Krzysztof Palczewski ◽

...

Keyword(s):

Outer Segment ◽

Molecular Mechanisms ◽

Structural Information ◽

Structural Basis ◽

Radius Of Curvature ◽

Rod Photoreceptor ◽

Molecular Architecture ◽

Structural Determinants ◽

Rod Outer Segment ◽

Wide Range

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkable long range order of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data reveal the existence of two molecular connectors/spacers which likely contribute to the nanometer scale precise stacking of the ROS disks. We further show that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) tetramers. We suggest that, together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Cryo-ET therefore provides novel quantitative and structural information on the molecular architecture in ROS and insights into possible mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

Download Full-text

Molecular dynamics of C99-bound γ-secretase reveal two binding modes with distinct compactness, stability, and active-site retention: implications for Aβ production

Biochemical Journal ◽

10.1042/bcj20190023 ◽

2019 ◽

Vol 476 (7) ◽

pp. 1173-1189 ◽

Cited By ~ 12

Author(s):

Budheswar Dehury ◽

Ning Tang ◽

Kasper P. Kepp

Keyword(s):

Structural Basis ◽

Binding Modes ◽

Wild Type ◽

Dynamic Simulations ◽

Aβ Peptides ◽

Cryo Electron Microscopy ◽

Compact State ◽

Β Sheet ◽

Aβ Production

Abstract The membrane protease γ-secretase cleaves the C99 fragment of the amyloid precursor protein, thus producing the Aβ peptides central to Alzheimer's disease. Cryo-electron microscopy has provided the topology but misses the membrane and loop parts that contribute to substrate binding. We report here an essentially complete atomic model of C99 within wild-type γ-secretase that respects all the experimental constraints and additionally describes loop, helix, and C99 substrate dynamics in a realistic all-atom membrane. Our model represents the matured auto-cleaved state required for catalysis. From two independent 500-ns molecular dynamic simulations, we identify two conformation states of C99 in equilibrium, a compact and a loose state. Our simulations provide a basis for C99 processing and Aβ formation and explain the production of longer and shorter Aβ, as the compact state retains C99 for longer and thus probably trims to shorter Aβ peptides. We expect pathogenic presenilin mutations to stabilize the loose over the compact state. The simulations detail the role of the Lys53–Lys54–Lys55 anchor for C99 binding, a loss of helicity of bound C99, and positioning of Thr48 and Leu49 leading to alternative trimming pathways on opposite sides of the C99 helix in three amino acid steps. The C99 binding topology resembles that of C83-bound γ-secretase without membrane but lacks a presenilin 1-C99 β-sheet, which could be induced by C83's stronger binding. The loose state should be selectively disfavored by γ-secretase modulators to increase C99 trimming and reduce the formation of longer Aβ, a strategy that is currently much explored but has lacked a structural basis.

Download Full-text

Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471400947x ◽

2014 ◽

Vol 70 (7) ◽

pp. 1944-1953 ◽

Cited By ~ 35

Author(s):

Eunha Hwang ◽

Hae-Kap Cheong ◽

Ameeq Ul Mushtaq ◽

Hye-Yeon Kim ◽

Kwon Joo Yeo ◽

...

Keyword(s):

Structural Information ◽

Critical Role ◽

Three Dimensional ◽

Helical Structure ◽

Structural Features ◽

Signalling Pathway ◽

Structural Basis ◽

X Ray Crystallography ◽

Hippo Signalling

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1–RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1–RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432–Lys437), which correspond to the short N-terminal 310-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1–RASSF5 complex showed a longer helical structure (Ser438–Lys480) than that in the MST1 homodimer (Val441–Lys480). Moreover, extensive polar and nonpolar contacts in the MST1–RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST–RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1–RASSF5 SARAH domain in apoptosis signalling.

Download Full-text

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

eLife ◽

10.7554/elife.07432 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 9

Author(s):

Agata Butryn ◽

Jan M Schuller ◽

Gabriele Stoehr ◽

Petra Runge-Wollmann ◽

Friedrich Förster ◽

...

Keyword(s):

Structural Information ◽

Structural Basis ◽

Independent Manner ◽

Histone Fold ◽

Structural Framework ◽

Wide Range ◽

Transcription Complexes ◽

Tata Box Binding Protein ◽

Structural Mechanisms ◽

Single Subunit

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.

Download Full-text

Determinants shaping the nanoscale architecture of the mouse rod outer segment

eLife ◽

10.7554/elife.72817 ◽

2021 ◽

Vol 10 ◽

Author(s):

Matthias Pöge ◽

Julia Mahamid ◽

Sanae S Imanishi ◽

Jürgen M Plitzko ◽

Krzysztof Palczewski ◽

...

Keyword(s):

Outer Segment ◽

Molecular Mechanisms ◽

Structural Information ◽

Structural Basis ◽

Radius Of Curvature ◽

Rod Photoreceptor ◽

Molecular Architecture ◽

Structural Determinants ◽

Rod Outer Segment ◽

Wide Range

The unique membrane organization of the rod outer segment (ROS), the specialized sensory cilium of rod photoreceptor cells, provides the foundation for phototransduction, the initial step in vision. ROS architecture is characterized by a stack of identically shaped and tightly packed membrane disks loaded with the visual receptor rhodopsin. A wide range of genetic aberrations have been reported to compromise ROS ultrastructure, impairing photoreceptor viability and function. Yet, the structural basis giving rise to the remarkably precise arrangement of ROS membrane stacks and the molecular mechanisms underlying genetically inherited diseases remain elusive. Here, cryo-electron tomography (cryo-ET) performed on native ROS at molecular resolution provides insights into key structural determinants of ROS membrane architecture. Our data confirm the existence of two previously observed molecular connectors/spacers which likely contribute to the nanometer-scale precise stacking of the ROS disks. We further provide evidence that the extreme radius of curvature at the disk rims is enforced by a continuous supramolecular assembly composed of peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) oligomers. We suggest that together these molecular assemblies constitute the structural basis of the highly specialized ROS functional architecture. Our Cryo-ET data provide novel quantitative and structural information on the molecular architecture in ROS and substantiate previous results on proposed mechanisms underlying pathologies of certain PRPH2 mutations leading to blindness.

Download Full-text

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

10.1101/2020.04.21.052910 ◽

2020 ◽

Cited By ~ 6

Author(s):

Kangcheng Song ◽

Miao Wei ◽

Wenjun Guo ◽

Yunlu Kang ◽

Jing-Xiang Wu ◽

...

Keyword(s):

Ion Channel ◽

Structural Information ◽

Structural Basis ◽

Binding Modes ◽

Inhibitor Binding ◽

Design And Optimization ◽

Inhibitory Function ◽

Physiological Processes ◽

Channel Inhibition ◽

Binding Pockets

AbstractTRPC5 channel is a non-selective cation channel that participates diverse physiological processes. Human TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression and kidney disease. Despite the high relevance of TRPC5 to human health, its inhibitor binding pockets have not been fully characterized due to the lack of structural information, which greatly hinders structure-based drug discovery. Here we show cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 Å. Based on the high-quality cryo-EM maps, we uncover the different binding pockets and detailed binding modes for these two inhibitors. Clemizole binds inside the voltage sensor-like domain of each subunit, while HC-070 binds close to the ion channel pore and is wedged between adjacent subunits. Both of them exert the inhibitory function by stabilizing the ion channel in a closed state. These structures provide templates for further design and optimization of inhibitors targeting human TRPC5.

Download Full-text

Cyclin-Dependent Kinase 2 in Cellular Senescence and Cancer. A Structural and Functional Review

Current Drug Targets ◽

10.2174/1389450120666181204165344 ◽

2019 ◽

Vol 20 (7) ◽

pp. 716-726 ◽

Cited By ~ 23

Author(s):

Priscylla Andrade Volkart ◽

Gabriela Bitencourt-Ferreira ◽

André Arigony Souto ◽

Walter Filgueira de Azevedo

Keyword(s):

Binding Affinity ◽

Cellular Senescence ◽

Cell Cycle Progression ◽

Structural Information ◽

Structural Basis ◽

Cyclin Dependent Kinase ◽

New Era ◽

Functional Studies ◽

Molecular Aspects

Background: Cyclin-dependent kinase 2 (CDK2) has been studied due to its role in the cell-cycle progression. The elucidation of the CDK2 structure paved the way to investigate the molecular basis for inhibition of this enzyme, with the coordinated efforts combining crystallography with functional studies. Objective: Our goal here is to review recent functional and structural studies directed to understanding the role of CDK2 in cancer and senescence. Methods: There are over four hundreds of crystallographic structures available for CDK2, many of them with binding affinity information. We use this abundance of data to analyze the essential features responsible for the inhibition of CDK2 and its function in cancer and senescence. Results: The structural and affinity data available CDK2 makes it possible to have a clear view of the vital CDK2 residues involved in molecular recognition. A detailed description of the structural basis for ligand binding is of pivotal importance in the design of CDK2 inhibitors. Our analysis shows the relevance of the residues Leu 83 and Asp 86 for binding affinity. The recent findings revealing the participation of CDK2 inhibition in senescence open the possibility to explore the richness of structural and affinity data for a new era in the development of CDK2 inhibitors, targeting cellular senescence. Conclusion: Here, we analyzed structural information for CDK2 in combination with inhibitors and mapped the molecular aspects behind the strongest CDK2 inhibitors for which structures and ligandbinding affinity data were available. From this analysis, we identified the significant intermolecular interactions responsible for binding affinity. This knowledge may guide the future development of CDK2 inhibitors targeting cancer and cellular senescence.

Download Full-text