scholarly journals Structural basis of Dscam1 homodimerization: Insights into context constraint for protein recognition

2016 ◽  
Vol 2 (5) ◽  
pp. e1501118 ◽  
Author(s):  
Shu-Ang Li ◽  
Linna Cheng ◽  
Yamei Yu ◽  
Jia-huai Wang ◽  
Qiang Chen
Keyword(s):  
Rna Splicing ◽  
Structural Information ◽  
Binding Specificity ◽  
Binding Mode ◽  
Structural Basis ◽  
Protein Recognition ◽  
Homophilic Binding ◽  
Symmetry Center ◽  
Cell Adhesion Molecule 1

The Drosophila neural receptor Dscam1 (Down syndrome cell adhesion molecule 1) plays an essential role in neuronal wiring and self-avoidance. Dscam1 potentially encodes 19,008 ectodomains through alternative RNA splicing and exhibits exquisite isoform-specific homophilic binding, which makes it an exceptional example for studying protein binding specificity. However, structural information on Dscam1 is limited, which hinders illumination of the mechanism of Dscam1 isoform-specific recognition. Whether different Dscam1 isoforms adopt the same dimerization mode remains a subject of debate. We present 12 Dscam1 crystal structures, provide direct evidence indicating that all isoforms adopt a conserved homodimer geometry in a modular fashion, identify two mechanisms for the Ig2 binding domain to dispel electrostatic repulsion during dimerization, decode Ig2 binding specificity by a central motif at its symmetry center, uncover the role of glycosylation in Dscam1 homodimerization, and find electrostatic potential complementarity to help define the binding region and the antiparallel binding mode. We then propose a concept that the context of a protein may set restrictions to regulate its binding specificity, which provides a better understanding of protein recognition.

scholarly journals Protein production, crystallization and preliminary crystallographic analysis of the four N-terminal immunoglobulin domains of Down syndrome cell adhesion molecule 1

2015 ◽  
Vol 71 (6) ◽  
pp. 775-778 ◽  
Author(s):  
Linna Cheng ◽  
Shu-Ang Li ◽  
Yamei Yu ◽  
Qiang Chen
Keyword(s):  
Down Syndrome ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Rna Splicing ◽  
Cell Adhesion Molecule ◽  
Crystallographic Analysis ◽  
Homophilic Binding ◽  
Ig Superfamily ◽  
Cell Adhesion Molecule 1 ◽  
Insight Into

Down syndrome cell adhesion molecule 1 (Dscam1), a member of the immunoglobulin (Ig) superfamily, plays important roles in both the nervous and the immune systems. Via alternative RNA splicing,DrosophilaDscam1 encodes a vast family of Ig-containing proteins that exhibit isoform-specific homophilic binding. Whether different Dscam1 isoforms adopt the same dimerization mode is under debate, and the detailed mechanism of Dscam1 specificity remains unclear. In this study, eight different isforms of Dscam1 Ig1–4 have been cloned, overexpressed, purified to homogeneity and crystallized. X-ray data were collected to 1.9–4.0 Å resolution. These structures will provide the opportunity to perform extensive structural comparisons of different Dscam1 isoforms and provide insight into its specificity.

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

2018 ◽  
Vol 115 (12) ◽  
pp. E2706-E2715 ◽  
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.

scholarly journals Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit K. Gandhi ◽  
Zhen-Yu J. Sun ◽  
Walter M. Kim ◽  
Yu-Hwa Huang ◽  
Yasuyuki Kondo ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Nmr Assignment ◽  
Structural Information ◽  
Microbial Pathogens ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Related Cell ◽  
Differential Scanning Fluorimetry ◽  
Cell Adhesion Molecule 1

AbstractHuman (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5, hTIM-3, PD-1, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC′ face including V39, I91, N97, and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A mutant monomer. These studies reveal the flexibility of the GFCC’ face and its important role in governing the formation of hCEACAM1 dimers and selective heterodimers.

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

2014 ◽  
Vol 70 (7) ◽  
pp. 1944-1953 ◽  
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.

scholarly journals Structural basis for regiospecific midazolam oxidation by human cytochrome P450 3A4

2016 ◽  
Vol 114 (3) ◽  
pp. 486-491 ◽  
Author(s):  
Irina F. Sevrioukova ◽  
Thomas L. Poulos
Keyword(s):  
Crystal Structure ◽  
Cytochrome P450 ◽  
Active Site ◽  
Structural Information ◽  
Binding Mode ◽  
Structural Basis ◽  
Cytochrome P450 3A4 ◽  
Human Cytochrome ◽  
Cyp3a4 Substrate

Human cytochrome P450 3A4 (CYP3A4) is a major hepatic and intestinal enzyme that oxidizes more than 60% of administered therapeutics. Knowledge of how CYP3A4 adjusts and reshapes the active site to regioselectively oxidize chemically diverse compounds is critical for better understanding structure–function relations in this important enzyme, improving the outcomes for drug metabolism predictions, and developing pharmaceuticals that have a decreased ability to undergo metabolism and cause detrimental drug–drug interactions. However, there is very limited structural information on CYP3A4–substrate interactions available to date. Despite the vast variety of drugs undergoing metabolism, only the sedative midazolam (MDZ) serves as a marker substrate for the in vivo activity assessment because it is preferentially and regioselectively oxidized by CYP3A4. We solved the 2.7 Å crystal structure of the CYP3A4–MDZ complex, where the drug is well defined and oriented suitably for hydroxylation of the C1 atom, the major site of metabolism. This binding mode requires H-bonding to Ser119 and a dramatic conformational switch in the F–G fragment, which transmits to the adjacent D, E, H, and I helices, resulting in a collapse of the active site cavity and MDZ immobilization. In addition to providing insights on the substrate-triggered active site reshaping (an induced fit), the crystal structure explains the accumulated experimental results, identifies possible effector binding sites, and suggests why MDZ is predominantly metabolized by the CYP3A enzyme subfamily.

scholarly journals Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium

2020 ◽  
Author(s):  
Amit K. Gandhi ◽  
Zhen-Yu J. Sun ◽  
Walter M. Kim ◽  
Yu-Hwa Huang ◽  
Yasuyuki Kondo ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Nmr Assignment ◽  
Structural Information ◽  
Microbial Pathogens ◽  
Structural Basis ◽  
X Ray ◽  
X Ray Crystallography ◽  
Related Cell ◽  
Differential Scanning Fluorimetry ◽  
Cell Adhesion Molecule 1

AbstractHuman (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5 and hTIM-3, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC’ face including V39, I91, N97 and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A monomer. These studies reveal the flexibility of the GFCC’ face and its important role in governing the formation of hCEACAM1 dimers and potentially heterodimers.

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

2021 ◽  
Vol 118 (6) ◽  
pp. e2010644118
Author(s):  
Rajasree Kalagiri ◽  
Robyn L. Stanfield ◽  
Jill Meisenhelder ◽  
James J. La Clair ◽  
Stephen R. Fuhs ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Information ◽  
Phosphate Group ◽  
Structural Basis ◽  
Binding Modes ◽  
Peptide Backbone ◽  
Wide Range ◽  
Complementarity Determining Regions ◽  
Histidine Phosphorylation

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.

scholarly journals Diversification by CofC and control by CofD govern biosynthesis and evolution of coenzyme F420 and its derivative 3PG-F420

2021 ◽  
Author(s):  
Mahmudul Hasan ◽  
Sabrina Schulze ◽  
Leona Berndt ◽  
Gottfried J Palm ◽  
Daniel Braga ◽  
...  
Keyword(s):  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Substrate Selection ◽  
Substrate Channeling ◽  
Biotechnological Production ◽  
Evolutionary Trajectory ◽  
And Control ◽  
Selection Of

Coenzyme F420 is a microbial redox cofactor that is increasingly used for biocatalytic applications. Recently, diversified biosynthetic routes to F420 and the discovery of a derivative, 3PG-F420, were reported. 3PG-F420 is formed via activation of 3-phospho-d-glycerate (3-PG) by CofC, but the structural basis of substrate binding, its evolution, as well as the role of CofD in substrate selection remained elusive. Here, we present a crystal structure of the 3-PG-activating CofC from Mycetohabitans sp. B3 and define amino acids governing substrate specificity. Site-directed mutagenesis enabled bidirectional switching of specificity and thereby revealed the short evolutionary trajectory to 3PG-F420 formation. Furthermore, CofC stabilized its product, thus confirming the structure of the unstable molecule, revealing its binding mode and suggesting a substrate channeling mechanism to CofD. The latter enzyme was shown to significantly contribute to the selection of related intermediates to control the specificity of the combined biosynthetic CofC/D step. Taken together, this work closes important knowledge gaps and opens up perspectives for the discovery, enhanced biotechnological production, and engineering of coenzyme F420 derivatives in the future.

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

2019 ◽  
Vol 20 (7) ◽  
pp. 716-726 ◽  
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

<P>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. </P><P> Objective: Our goal here is to review recent functional and structural studies directed to understanding the role of CDK2 in cancer and senescence. </P><P> 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. </P><P> 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. </P><P> 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.</P>

Structural Basis for Effector Control and Redox Partner Recognition in Cytochrome P450

Science ◽  
2013 ◽  
Vol 340 (6137) ◽  
pp. 1227-1230 ◽  
Author(s):  
Sarvind Tripathi ◽  
Huiying Li ◽  
Thomas L. Poulos
Keyword(s):  
Crystal Structure ◽  
Electron Transfer ◽  
Structural Information ◽  
Cytochromes P450 ◽  
Structural Basis ◽  
Proton Coupled Electron Transfer ◽  
Redox Partner ◽  
Open Conformation ◽  
Redox Partners

Cytochromes P450 catalyze a variety of monooxygenase reactions that require electron transfer from redox partners. Although the structure of many P450s and a small handful of redox partners are known, there is very little structural information available on redox complexes, thus leaving a gap in our understanding on the control of P450–redox partner interactions. We have solved the crystal structure of oxidized and reduced P450cam complexed with its redox partner, putidaredoxin (Pdx), to 2.2 and 2.09 angstroms, respectively. It was anticipated that Pdx would favor closed substrate-bound P450cam, which differs substantially from the open conformer, but instead we found that Pdx favors the open state. These new structures indicate that the effector role of Pdx is to shift P450cam toward the open conformation, which enables the establishment of a water-mediated H-bonded network, which is required for proton-coupled electron transfer.

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