Mutational and biophysical robustness in a prestabilized monobody

Chandler, Peter G.; Tan, Li Lynn; Porebski, Benjamin T.; Green, James S.; Riley, Blake T.; Broendum, Sebastian S.; Hoke, David E.; Falconer, Robert J.; Munro, Trent P.; Buckle, Malcolm; Jackson, C.J.; Buckle, Ashley M.

doi:10.1016/j.jbc.2021.100447

Cited by 10 publications

(8 citation statements)

References 71 publications

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“…Kd values of CRT3-Rluc8 and CRT4-Rluc8 (7.990 ± 0.870 nM and 8.686 ± 0.836 nM) showed efficient CRT binding ( Figure 3 C). These values were comparable to the affinities of other monobody proteins and typical antibodies [ 64 , 65 , 66 , 67 , 68 ], further demonstrating that the engineered CRT3 and CRT4 monobodies had strong binding affinities for CRT.…”

Section: Resultssupporting

confidence: 63%

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Zhang

Thangam

You

et al. 2021

Cancers

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Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

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Section: Resultssupporting

confidence: 63%

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Zhang

Thangam

You

et al. 2021

Cancers

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“…Since its introduction in the 1990s, the FN3 scaffold has been modified with the goal of enhancing its stability so that it can accept an ever greater number and diversity of sequence variants ( 43 , 44 , 45 ). Using one of the ultra-stable FN3 derivatives as a starting scaffold might permit isolation of a wider range of sequence variants than was observed in the present study.…”

Section: Discussionmentioning

confidence: 99%

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

Zhu,

Smallwood,

Rattner

et al. 2024

Journal of Biological Chemistry

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“…Some binding energy is inevitably siphoned off to drive the fold shift. The main reason, however is that transposing the ligand-contacting residues from different monobodies onto a common scaffold does not capture all the nuances of the binding interactions 38,53 . This method can reproduce parental binding affinities (e.g., FN3 SUMO (Table 2) and a VEGFR2-binding consensus-designed FN3 53 ), but these cases seem to be exceptions.…”

Section: Discussionmentioning

confidence: 99%

“…The main reason, however is that transposing the ligand-contacting residues from different monobodies onto a common scaffold does not capture all the nuances of the binding interactions 38,53 . This method can reproduce parental binding affinities (e.g., FN3 SUMO (Table 2) and a VEGFR2-binding consensus-designed FN3 53 ), but these cases seem to be exceptions. Additional computational redesign, such as that employed in the lucCage study, may be expected to improve affinity.…”

Section: Discussionmentioning

confidence: 99%

An adaptable, monobody-based biosensor scaffold with FRET output

Presti

Loh

2022

Preprint

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Protein-based fluorescent biosensors are powerful tools for analyte recognition in vitro and in cells. Numerous proteinaceous binding scaffolds have been developed that recognize ligands with affinity and specificity comparable to those of conventional antibodies, but are smaller, readily overexpressed, and more amenable to engineering. Like antibodies, these binding domains are useful as recognition modules in protein switches and biosensors, but they are not capable of reporting on the binding event by themselves. Here, we engineer a small binding scaffold—a consensus-designed fibronectin 3 monobody—such that it undergoes a conformational change upon ligand binding. This change is detected by Förster resonance energy transfer using chemical dyes or cyan and yellow fluorescent proteins as donor/acceptor groups. By grafting substrate recognition residues from different monobodies onto this scaffold, we create fluorescent biosensors for c-Abl Src homology 2 (SH2) domain, WD40-repeat protein 5 (WDR5), small ubiquitin-like modifier-1 (SUMO), and h-Ras. The biosensors bind their cognate ligands reversibly, with affinities consistent with those of the parent monobodies, and with half times of seconds to minutes. This design serves as generalizable platform for creating a genetically-encoded, ratiometric biosensors by swapping binding residues from known monobodies, with minimal modification.

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Mutational and biophysical robustness in a prestabilized monobody

Cited by 10 publications

References 71 publications

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Utility of protein–protein binding surfaces composed of anti-parallel alpha-helices and beta-sheets selected by phage display

An adaptable, monobody-based biosensor scaffold with FRET output

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