Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

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Validation and Stabilization of a Prophage Lysin of Clostridium perfringens by Using Yeast Surface Display and Coevolutionary Models

Applied and Environmental Microbiology ◽

10.1128/aem.00054-19 ◽

2019 ◽

Vol 85 (10) ◽

Cited By ~ 3

Author(s):

Seth C. Ritter ◽

Benjamin J. Hackel

Keyword(s):

Protein Stability ◽

Clostridium Perfringens ◽

High Throughput Sequencing ◽

Surface Display ◽

High Specificity ◽

Classification Performance ◽

Yeast Surface Display ◽

Content Type ◽

Cell Wall Binding ◽

Yeast Surface

ABSTRACT Bacteriophage lysins are compelling antimicrobial proteins whose biotechnological utility and evolvability would be aided by elevated stability. Lysin catalytic domains, which evolved as modular entities distinct from cell wall binding domains, can be classified into one of several families with highly conserved structure and function, many of which contain thousands of annotated homologous sequences. Motivated by the quality of these evolutionary data, the performance of generative protein models incorporating coevolutionary information was analyzed to predict the stability of variants in a collection of 9,749 multimutants across 10 libraries diversified at different regions of a putative lysin from a prophage region of a Clostridium perfringens genome. Protein stability was assessed via a yeast surface display assay with accompanying high-throughput sequencing. Statistical fitness of mutant sequences, derived from second-order Potts models inferred with different levels of sequence homolog information, was predictive of experimental stability with areas under the curve (AUCs) ranging from 0.78 to 0.85. To extract an experimentally derived model of stability, a logistic model with site-wise score contributions was regressed on the collection of multimutants. This achieved a cross-validated classification performance of 0.95. Using this experimentally derived model, 5 designs incorporating 5 or 6 mutations from multiple libraries were constructed. All designs retained enzymatic activity, with 4 of 5 increasing the melting temperature and with the highest-performing design achieving an improvement of +4°C. IMPORTANCE Bacteriophage lysins exhibit high specificity and activity toward host bacteria with which the phage coevolved. These properties of lysins make them attractive for use as antimicrobials. Although there has been significant effort to develop platforms for rapid lysin engineering, there have been numerous shortcomings when pursuing the ultrahigh throughput necessary for the discovery of rare combinations of mutations to improve performance. In addition to validation of a putative lysin and stabilization thereof, the experimental and computational methods presented here offer a new avenue for improving protein stability and are easily scalable to analysis of tens of millions of mutations in single experiments.

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Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution

Journal of Biological Chemistry ◽

10.1074/jbc.m116.756718 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3481-3495 ◽

Cited By ~ 30

Author(s):

Valeria Arkadash ◽

Gal Yosef ◽

Jason Shirian ◽

Itay Cohen ◽

Yuval Horev ◽

...

Keyword(s):

Directed Evolution ◽

Surface Display ◽

High Specificity ◽

Computational Design ◽

Computational Method ◽

Yeast Surface Display ◽

Mmp Inhibitor ◽

Starting Point ◽

Yeast Surface

Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.

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Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer

Cancers ◽

10.3390/cancers12102824 ◽

2020 ◽

Vol 12 (10) ◽

pp. 2824

Author(s):

Ron Amon ◽

Ronit Rosenfeld ◽

Shahar Perlmutter ◽

Oliver C. Grant ◽

Sharon Yehuda ◽

...

Keyword(s):

Cancer Therapy ◽

Therapeutic Efficacy ◽

Surface Display ◽

Yeast Surface Display ◽

Structural Basis ◽

Therapeutic Antibodies ◽

Pancreatic Cancers ◽

Yeast Surface ◽

Directed Molecular Evolution ◽

Cancer Theranostics

Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19−9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.

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Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display

Nucleic Acids Research ◽

10.1093/nar/gnh030 ◽

2004 ◽

Vol 32 (3) ◽

pp. 36e-36 ◽

Cited By ~ 66

Author(s):

J. S. Swers

Keyword(s):

Homologous Recombination ◽

Surface Display ◽

Yeast Surface Display ◽

Antibody Libraries ◽

Yeast Surface

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Structural basis for adhesion G protein-coupled receptor Gpr126 function

10.1101/805515 ◽

2019 ◽

Author(s):

Katherine Leon ◽

Rebecca L. Cunningham ◽

Joshua A. Riback ◽

Ezra Feldman ◽

Jingxian Li ◽

...

Keyword(s):

Binding Site ◽

G Protein ◽

Heart Development ◽

Calcium Binding ◽

Structural Basis ◽

Calcium Binding Site ◽

Surface Receptors ◽

Alternatively Spliced ◽

G Protein Coupled

AbstractMany drugs target the extracellular regions (ECRs) of cell-surface receptors. The large and alternatively-spliced ECRs of adhesion G protein-coupled receptors (aGPCRs) have key functions in diverse biological processes including neurodevelopment, embryogenesis, and tumorigenesis. However, their structures and mechanisms of action remain unclear, hampering drug development. The aGPCR Gpr126/Adgrg6 regulates Schwann cell myelination, ear canal formation, and heart development; and GPR126 mutations cause myelination defects in human. Here, we determine the structure of the complete zebrafish Gpr126 ECR and reveal five domains including a previously-unknown domain. Strikingly, the Gpr126 ECR adopts a closed conformation that is stabilized by an alternatively spliced linker and a conserved calcium-binding site. Alternative splicing regulates ECR conformation and receptor signaling, while mutagenesis of the newly-characterized calcium-binding site abolishes Gpr126 function in vivo. These results demonstrate that Gpr126 ECR utilizes a multi-faceted dynamic approach to regulate receptor function and provide novel insights into ECR-targeted drug design.

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Structural Basis for Cytoplasmic Dynein-1 Regulation by Lis1

10.1101/2021.06.11.448119 ◽

2021 ◽

Author(s):

John P Gillies ◽

Janice M Reimer ◽

Eva P Karasmanis ◽

Indrajit Lahiri ◽

Zaw Min Htet ◽

...

Keyword(s):

High Resolution ◽

Cytoplasmic Dynein ◽

Motor Domain ◽

Structural Basis ◽

Neurodevelopmental Disease ◽

Microtubule Motor ◽

High Resolution Structure ◽

Resolution Structure

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator that binds directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's β-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.

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Structural basis for adhesion G protein-coupled receptor Gpr126 function

Nature Communications ◽

10.1038/s41467-019-14040-1 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 6

Author(s):

Katherine Leon ◽

Rebecca L. Cunningham ◽

Joshua A. Riback ◽

Ezra Feldman ◽

Jingxian Li ◽

...

Keyword(s):

Binding Site ◽

G Protein ◽

Heart Development ◽

Calcium Binding ◽

Structural Basis ◽

Calcium Binding Site ◽

Surface Receptors ◽

Alternatively Spliced ◽

G Protein Coupled

AbstractMany drugs target the extracellular regions (ECRs) of cell-surface receptors. The large and alternatively-spliced ECRs of adhesion G protein-coupled receptors (aGPCRs) have key functions in diverse biological processes including neurodevelopment, embryogenesis, and tumorigenesis. However, their structures and mechanisms of action remain unclear, hampering drug development. The aGPCR Gpr126/Adgrg6 regulates Schwann cell myelination, ear canal formation, and heart development; and GPR126 mutations cause myelination defects in human. Here, we determine the structure of the complete zebrafish Gpr126 ECR and reveal five domains including a previously unknown domain. Strikingly, the Gpr126 ECR adopts a closed conformation that is stabilized by an alternatively spliced linker and a conserved calcium-binding site. Alternative splicing regulates ECR conformation and receptor signaling, while mutagenesis of the calcium-binding site abolishes Gpr126 function in vivo. These results demonstrate that Gpr126 ECR utilizes a multi-faceted dynamic approach to regulate receptor function and provide relevant insights for ECR-targeted drug design.

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Structural basis of cowpox evasion of NKG2D immunosurveillance

10.1101/796862 ◽

2019 ◽

Author(s):

Eric Lazear ◽

Michel M. Sun ◽

Xiaoli Wang ◽

Theresa L. Geurs ◽

Christopher A. Nelson ◽

...

Keyword(s):

Cell Surface ◽

Surface Display ◽

Orphan Receptor ◽

Structural Basis ◽

Zoonotic Potential ◽

Cowpox Virus ◽

Nkg2d Ligands ◽

Binding Orientation ◽

Deficient Mice

AbstractNKG2D is a key component of cytotoxic antitumor and antiviral responses. Multiple viruses evade NKG2D recognition by blocking NKG2D ligand expression on infected cells. In contrast, cowpox virus targets NKG2D directly by encoding a secreted antagonist, Orthopoxvirus MHC Class I-like Protein (OMCP). We have previously reported that OMCP also binds to the orphan receptor FcRL5 on innate B cells. Here, we demonstrate that mammalian-derived, glycosylated OMCP binds NKG2D but not FcRL5. Cowpox viruses either lacking OMCP, or expressing an NKG2D-binding deficient mutant, are significantly attenuated in wild type and FcRL5-deficient mice but not NKG2D-deficient mice, demonstrating that OMCP is critical in subverting NKG2D-mediated immunity in vivo. Next we determined the structure of OMCP bound to human NKG2D. Despite a structure similar to that of host NKG2D ligands, OMCP uses a drastically different orientation for NKG2D binding. The re-orientation of OMCP is associated with dramatically higher affinity for human NKG2D and the targeted interface is highly conserved in mammalian NKG2Ds, increasing the zoonotic potential of cowpox virus. We also show that cell surface presented OMCP can trigger NKG2D effector functions equivalently to host NKG2D ligands, demonstrating that NKG2D-mediated signaling requires clustering but is insensitive to binding orientation. Thus, in contrast to TCR/MHC interactions, the docking topology of NKG2D with its ligands does not appear to regulate its activation.Author SummaryVirally infected or tumor-transformed cells display NKG2D ligands (NKG2DLs) on their cell surface, which activates NKG2D-bearing lymphocytes to kill the transformed cell. Pathogens are known to counter this by blocking NKG2DL expression and/or surface display. In contrast, some tumor cells cleave endogenous NKG2DLs creating soluble NKG2D antagonists. Unlike other viral pathogens, cowpox virus uses a strategy analogous to cancer cells by targeting NKG2D directly with a soluble, high affinity NKG2D-antagonist named OMCP. We determined that OMCP’s virulence in vivo is attributed to blocking NKG2D-mediated NK cell responses with no apparent effect due to binding to other receptors or cell types. We have also determined the crystal structure of cowpox OMCP bound to human NKG2D, revealing that despite conservation of the ligand scaffolding with host NKG2DLs, the viral protein is engaged with a radically altered orientation compared to all host NKG2DLs. Our structure provides key insight into how OMCP binds with an ∼5,000-fold increased affinity compared to human NKG2DLs and show that the OMCP binding site is exceptionally conserved among primates and rodents, suggesting that the ability of OMCP to recognize this conserved interface contributes to the broad zoonotic potential of cowpox virus. Finally, we show that cell membrane-anchored OMCP can trigger equivalent NKG2D-mediated killing as host NKG2DLs, demonstrating that NKG2D signaling is insensitive to ligand binding orientation.

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Targeted DNA transposition using a dCas9-transposase fusion protein

10.1101/571653 ◽

2019 ◽

Author(s):

Shivam Bhatt ◽

Ronald Chalmers

Keyword(s):

Fusion Protein ◽

Binding Site ◽

Biochemical Analysis ◽

Genome Engineering ◽

High Specificity ◽

Target Dna ◽

Nucleoprotein Complexes ◽

Significant Conformational Change

SUMMARYHomology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

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Structural basis for cytoplasmic dynein-1 regulation by Lis1

eLife ◽

10.7554/elife.71229 ◽

2022 ◽

Vol 11 ◽

Author(s):

John P Gillies ◽

Janice M Reimer ◽

Eva P Karasmanis ◽

Indrajit Lahiri ◽

Zaw Min Htet ◽

...

Keyword(s):

High Resolution ◽

Cytoplasmic Dynein ◽

Motor Domain ◽

Structural Basis ◽

Neurodevelopmental Disease ◽

Molecular Detail ◽

Microtubule Motor ◽

High Resolution Structure ◽

Resolution Structure

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein–Lis1 complex. Our 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.

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