Structural properties and peptide ligand binding of the capsid homology domains of human Arc

AbstractAllosteric regulation is an innate control in most metabolic and signalling cascades that enables living organisms to adapt to the changing environment by tuning the affinity and regulating the activity of target proteins. For a microscopic understanding of this process, a protein system has been designed in such a way that allosteric communication between the binding and allosteric site can be observed in both directions. To that end, an azobenzene-derived photoswitch has been linked to the α3-helix of the PDZ3 domain, arguably the smallest allosteric protein with a clearly identifiable binding and allosteric site. Photo-induced trans-to-cis isomerisation of the photoswitch increases the binding affinity of a small peptide ligand to the protein up to 120-fold, depending on temperature. At the same time, ligand binding speeds up the thermal cis-to-trans back-isomerisation rate of the photoswitch. Based on the energetics of the four states of the system (cis vs trans and ligand-bound vs free), the concept of an allosteric force is introduced, which can be used to drive chemical reactions.

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Structural studies of the phosphatidylinositol 3-kinase (PI3K) SH3 domain in complex with a peptide ligand: role of the anchor residue in ligand binding

Biological Chemistry ◽

10.1515/bc.2010.003 ◽

2010 ◽

Vol 391 (1) ◽

Cited By ~ 14

Author(s):

Renu Batra-Safferling ◽

Joachim Granzin ◽

Susanne Mödder ◽

Silke Hoffmann ◽

Dieter Willbold

Keyword(s):

Crystal Structure ◽

Ligand Binding ◽

Protein Interactions ◽

Intracellular Signaling ◽

Sh3 Domain ◽

Peptide Ligand ◽

Type I ◽

Phosphatidylinositol 3 Kinase ◽

Anchor Residue ◽

Phosphatidylinositol 3

Abstract Src homology 3 (SH3) domains are mediators of protein-protein interactions. They comprise approximately 60 amino acid residues and are found in many intracellular signaling proteins. Here, we present the crystal structure of the SH3 domain from phosphatidylinositol 3-kinase (PI3K) in complex with the 12-residue proline-rich peptide PD1R (HSKRPLPPLPSL). The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 Å reveals type I ligand orientation of the bound peptide with an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of the SH3 domain shows minimal changes on ligand binding. In addition, we also attempted crystallization with another peptide ligand (PD1) where the residue at anchor position P-3 is a tyrosine. The crystals obtained did not contain the PD1 ligand; instead, the ligand binding site is partially occupied by residues Arg18 and Trp55 from the symmetry-related PI3K SH3 molecule. Considering these crystal structures of PI3K SH3 together with published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify the individual residues which play an important role in defining target specificity.

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Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology

CrossRef Listing of Deleted DOIs ◽

10.1177/108705719900400407 ◽

1999 ◽

Vol 4 (4) ◽

pp. 193-204 ◽

Cited By ~ 54

Author(s):

Sheri Miraglia ◽

Elana E. Swartzman ◽

Julia Mellentin-Michelotti ◽

Lolita Evangelista ◽

Christopher Smith ◽

...

Keyword(s):

Ligand Binding ◽

High Throughput ◽

High Throughput Screening ◽

Laser Scanner ◽

Peptide Ligand ◽

Binding Assays ◽

Using Data ◽

Immunological Assays ◽

The Individual ◽

Homogeneous Assays

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm2 digitized image of a 100-μm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twister™ (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

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Affinity Enhancers for Peptide Ligand Binding to the Extracellular Domain of the Amylin Receptor 2

The FASEB Journal ◽

10.1096/fasebj.2021.35.s1.02326 ◽

2021 ◽

Vol 35 (S1) ◽

Author(s):

Sangmin Lee ◽

Augen Pioszak

Keyword(s):

Ligand Binding ◽

Extracellular Domain ◽

Peptide Ligand

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Structural Investigations on the SH3b Domains of Clostridium perfringens Autolysin through NMR Spectroscopy and Structure Simulation Enlighten the Cell Wall Binding Function

Molecules ◽

10.3390/molecules26185716 ◽

2021 ◽

Vol 26 (18) ◽

pp. 5716

Author(s):

Yubao Shan ◽

Xiaoling He ◽

Zi Wang ◽

Xiali Yue ◽

Jiang Zhu ◽

...

Keyword(s):

Cell Wall ◽

Nmr Spectroscopy ◽

Ligand Binding ◽

Clostridium Perfringens ◽

Catalytic Domain ◽

Peptide Ligand ◽

Structural Investigations ◽

Cell Wall Binding ◽

Potential Ligand ◽

And Function

Clostridium perfringens autolysin (CpAcp) is a peptidoglycan hydrolase associated with cell separation, division, and growth. It consists of a signal peptide, ten SH3b domains, and a catalytic domain. The structure and function mechanisms of the ten SH3bs related to cell wall peptidoglycan binding remain unclear. Here, the structures of CpAcp SH3bs were studied through NMR spectroscopy and structural simulation. The NMR structure of SH3b6 was determined at first, which adopts a typical β-barrel fold and has three potential ligand-binding pockets. The largest pocket containing eight conserved residues was suggested to bind with peptide ligand in a novel model. The structures of the other nine SH3bs were subsequently predicted to have a fold similar to SH3b6. Their ligand pockets are largely similar to those of SH3b6, although with varied size and morphology, except that SH3b1/2 display a third pocket markedly different from those in other SH3bs. Thus, it was supposed that SH3b3-10 possess similar ligand-binding ability, while SH3b1/2 have a different specificity and additional binding site for ligand. As an entirety, ten SH3bs confer a capacity for alternatively binding to various peptidoglycan sites in the cell wall. This study presents an initial insight into the structure and potential function of CpAcp SH3bs.

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