A Hot-Spot Motif Characterizes the Interface between a Designed Ankyrin-Repeat Protein and Its Target Ligand

Cheung, Luthur Siu Lun; Kanwar, Manu; Ostermeier, Marc; Κωνσταντόπουλος, Κωνσταντίνος

doi:10.1016/j.bpj.2012.01.004

Cited by 22 publications

(30 citation statements)

References 50 publications

(64 reference statements)

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“…We examined the effect of the linker length on target trapping in vitro by expressing Rsp5 UBAITs containing a GGSG flexible linker sequence [33,41,42] …”

Section: Characterization Of Rsp5 Ubaits In Vitromentioning

confidence: 99%

“…We examined the effect of the linker length on target trapping in vitro by expressing Rsp5 UBAITs containing a GGSG flexible linker sequence [33,41,42] repeated 1, 3, or 5 times, resulting in linker lengths of 4, 12, or 20 amino acids. As shown in Appendix Fig S2, all three WT UBAITs supported target trapping in vitro, with the longer linker lengths supporting more efficient conjugation to Wbp2.…”

Section: Characterization Of Rsp5 Ubaits In Vitromentioning

confidence: 99%

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Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

et al. 2015

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We describe a new class of reagents for identifying substrates, adaptors, and regulators of HECT and RING E3s. UBAITs (UbiquitinActivated Interaction Traps) are E3-ubiquitin fusion proteins and, in an E1-and E2-dependent manner, the C-terminal ubiquitin moiety forms an amide linkage to proteins that interact with the E3, enabling covalent co-purification of the E3 with partner proteins. We designed UBAITs for both HECT (Rsp5, Itch) and RING (Psh1, RNF126, RNF168) E3s. For HECT E3s, trapping of interacting proteins occurred in vitro either through an E3 thioester-linked lariat intermediate or through an E2 thioester intermediate, and both WT and active-site mutant UBAITs trapped known interacting proteins in yeast and human cells. Yeast Psh1 and human RNF126 and RNF168 UBAITs also trapped known interacting proteins when expressed in cells. Human RNF168 is a key mediator of ubiquitin signaling that promotes DNA double-strand break repair. Using the RNF168 UBAIT, we identify H2AZ-a histone protein involved in DNA repair-as a new target of this E3 ligase. These results demonstrate that UBAITs represent powerful tools for profiling a wide range of ubiquitin ligases.

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“…We examined the effect of the linker length on target trapping in vitro by expressing Rsp5 UBAITs containing a GGSG flexible linker sequence [33,41,42] …”

Section: Characterization Of Rsp5 Ubaits In Vitromentioning

confidence: 99%

Section: Characterization Of Rsp5 Ubaits In Vitromentioning

confidence: 99%

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

et al. 2015

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“…To obtain a surface that would readily bind soluble proteins, we silanized MFP cantilevers (Bruker Nano, Camarillo, CA) with 2% 3-aminopropyltriethoxysilane (13)(14)(15)22). The cantilevers were then incubated in a 5 mg/ml solution of fibrinogen in DPBS containing 50-fold molar excess of the cross-linker bis(sulfosuccinimidyl) suberate (BS 3 ; Pierce, Rockford, IL) for 30 min followed by quenching with Tris buffer.…”

Section: Cantilever Functionalizationmentioning

confidence: 99%

“…We conducted experiments using an MFP (Asylum Research, Santa Barbara, CA) (13)(14)(15)22). We calibrated the softest triangular cantilever (C type cantilever, MLCT probes) using the thermal noise amplitude and measured its deflection by laser reflection onto a split photodetector (Fig.…”

Section: Single-molecule Force Spectroscopy Data Acquisition and Anmentioning

confidence: 99%

Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

Raman

Alves

Wirtz

et al. 2012

Biophysical Journal

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CD44 is a multifunctional glycoprotein that binds to hyaluronan and fibrin(ogen). Alternative splicing is responsible for the generation of numerous different isoforms, the smallest of which is CD44s. Insertion of variant exons into the extracellular membrane proximal region generates the variant isoforms (CD44v). Here, we used force spectroscopy to delineate the biophysical and molecular requirements of CD44-HA and CD44-fibrin(ogen) interactions at the single-molecule level. CD44v-HA and CD44s-HA single bonds exhibit similar kinetic and micromechanical properties because the HA-binding motif on CD44 is common to all of the isoforms. Although this is the primary binding site, O- and N-linked glycans and sulfation also contribute to the tensile strength of the CD44-HA bond. The CD44s-fibrin pair has a lower unstressed dissociation rate and a higher tensile strength than CD44s-fibrinogen but is weaker than the CD44-HA bond. In contrast to CD44-HA binding, the molecular interaction between CD44 and fibrin(ogen) is predominantly mediated by the chondroitin sulfate and dermatan sulfate on CD44. Blocking sulfation on CD44s modestly decreases the tensile strength of CD44s-fibrin(ogen) binding, which is in stark contrast to CD44v-fibrin interaction. Collectively, the results obtained by force spectroscopy in conjunction with biochemical interventions enable us to delineate the biophysical parameters and molecular constituents of CD44 binding to hyaluronan and fibrin(ogen).

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“…In general, only a few important residues play a major role in protein-ligand binding (Burgoyne and Jackson, 2006;Barillari et al, 2008;Cheung et al, 2012;Bauman et al, 2016). Therefore, it is crucial to identify these key residues and explore the binding mechanism of protein-ligand to improve the binding potency and selectivity of ligand.…”

Section: Introductionmentioning

confidence: 99%

Molecular Mechanism of Selective Binding of NMS-P118 to PARP-1 and PARP-2: A Computational Perspective

Wang

Cong

et al. 2020

Front. Mol. Biosci.

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The nuclear protein poly (ADP-ribose) polymerase-1 (PARP-1) inhibitors have been proven effective to potentiate both chemotherapeutic agents and radiotherapy. However, a major problem of most current PARP inhibitors is their lack of selectivity for PARP-1 and its closest isoform PARP-2. NMS-P118 is a highly selective PARP inhibitor that binds PARP-1 stronger than PARP-2 and has many advantages such as excellent pharmacokinetic profiles. In this study, molecular dynamics (MD) simulations of NMS-P118 in complex with PARP-1 and PARP-2 were performed to understand the molecular mechanism of its selectivity. Alanine scanning together with free energy calculation using MM/GBSA and interaction entropy reveal key residues that are responsible for the selectivity. Although the conformation of the binding pockets and NMS-P118 are very similar in PARP-1 and PARP-2, most of the hot-spot residues in PARP-1 have stronger binding free energy than the corresponding residues in PARP-2. Detailed analysis of the binding energy shows that the 4 ′ 4-difluorocyclohexyl ring on NMS-P118 form favorable hydrophobic interaction with Y889 in PARP-1. In addition, the H862 residue in PARP-1 has stronger binding free energy than H428 in PARP-2, which is due to shorter distance and stronger hydrogen bonds. Moreover, the negatively charged E763 residue in PARP-1 forms stronger electrostatic interaction energy with the positively charged NMS-P118 than the Q332 residue in PARP-2. These results rationalize the selectivity of NMS-P118 and may be useful for designing novel selective PARP inhibitors.

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A Hot-Spot Motif Characterizes the Interface between a Designed Ankyrin-Repeat Protein and Its Target Ligand

Cited by 22 publications

References 50 publications

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

Ubiquitin‐Activated Interaction Traps ( UBAIT s) identify E3 ligase binding partners

Distinct Kinetic and Molecular Requirements Govern CD44 Binding to Hyaluronan versus Fibrin(ogen)

Molecular Mechanism of Selective Binding of NMS-P118 to PARP-1 and PARP-2: A Computational Perspective

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