Machine learning guided design of high affinity ACE2 decoys for SARS-CoV-2 neutralization

A potential therapeutic candidate for neutralizing SARS-CoV-2 infection is engineering high- affinity soluble ACE2 decoy proteins to compete for binding of the viral spike (S) protein. Previously, a deep mutational scan of ACE2 was performed and has led to the identification of a triple mutant ACE2 variant, named ACE22.v.2.4, that exhibits nanomolar affinity binding to the RBD domain of S. Using a recently developed transfer learning algorithm, TLmutation, we sought to identified other ACE2 variants, namely double mutants, that may exhibit similar binding affinity with decreased mutational load. Upon training a TLmutation model on the effects of single mutations, we identified several ACE2 double mutants that bind to RBD with tighter affinity as compared to the wild type, most notably, L79V;N90D that binds RBD with similar affinity to ACE22.v.2.4. The successful experimental validation of the double mutants demonstrated the use transfer and supervised learning approaches for engineering protein-protein interactions and identifying high affinity ACE2 peptides for targeting SARS-CoV-2.

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Mutation Effects on Remote Epitopes of An Autoantigen Decoded with Simulated B-Factors

10.1101/559963 ◽

2019 ◽

Author(s):

Yuan-Ping Pang ◽

Marta Casal Moura ◽

Gwen E. Thompson ◽

Amber M. Hummel ◽

Darlene R. Nelson ◽

...

Keyword(s):

Autoimmune Diseases ◽

Protein Interactions ◽

Granulomatosis With Polyangiitis ◽

Wild Type ◽

Protein Protein Interactions ◽

Triple Mutant ◽

Experimental Conditions ◽

Chain Flexibility

Proteinase 3 (PR3) is a neutrophil serine protease targeted by anti-neutrophil cytoplasmic antibodies (ANCAs) in the autoimmune disease granulomatosis with polyangiitis (GPA)1–5. PR3 mutants were developed to investigate how PR3 interacts with the ANCAs and whether the interactions can be intervened by therapeutics. One mutant with a Ser195Ala mutation (iPR3-Val103) recognized as many ANCAs as wild-type PR3 (PR3-Val103)6–9, indicating that PR3-Val103 and iPR3-Val103 have equivalent ANCA-binding capabilities. A triple mutant of the latter (iHm5-Val103) bound a monoclonal antibody (moANCA518) from a patient with GPA on an epitope remote from the mutation sites. Unexpectedly, the corresponding epitope of iPR3-Val103 was inaccessible to moANCA518 under the same experimental conditions10,11. These observations demonstrate that a latent epitope of PR3 can be activated surprisingly by remote mutations in PR311. Here we report a comparative analysis of simulated B-factors (i.e., measurements of local mobility) of PR3-Val103, iPR3-Val103, and iHm5-Val103, demonstrating that the binding of moANCA518 to iHm5-Val103 is enabled by an increase in main-chain flexibility in the latent epitope caused by the remote muta-tions in iHm5-Val103. This epitope activation—achieved in vitro by remote mutations as we demonstrated or in vivo conceivably by remote protein»protein interactions12 or remote po-lymorphisms—may be a fundamental feature of antibody-mediated autoimmune diseases. Rigidifying B-cell epitopes on an autoantigen with therapeutics designed using the B-factor analysis disclosed here may lead to effective treatments for these autoimmune diseases by making the autoantigen inaccessible to existing autoantibodies. This analysis may also be used to predict and characterize epitopes and remote mutation effects.

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Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

Scientific Reports ◽

10.1038/s41598-021-83265-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Avital Shushan ◽

Mickey Kosloff

Keyword(s):

Protein Interactions ◽

Structural Elements ◽

Protein Protein Interactions ◽

Immunity Protein ◽

Rational Engineering ◽

High Affinity ◽

Comparative Structure ◽

Polar Interactions ◽

Exquisite Specificity

AbstractThe interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein–protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1–α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1–α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein–protein interactions, with implications for drug development and rational engineering of these interfaces.

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PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

The Journal of Cell Biology ◽

10.1083/jcb.132.3.359 ◽

1996 ◽

Vol 132 (3) ◽

pp. 359-370 ◽

Cited By ~ 115

Author(s):

E F Smith ◽

P A Lefebvre

Keyword(s):

Protein Interactions ◽

Insertional Mutagenesis ◽

Flagellar Motility ◽

Wild Type ◽

Protein Protein Interactions ◽

Cellular Functions ◽

Central Pair ◽

Central Apparatus ◽

Immunofluorescence Labeling ◽

Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

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The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85

International Journal of Molecular Sciences ◽

10.3390/ijms21010125 ◽

2019 ◽

Vol 21 (1) ◽

pp. 125

Author(s):

Francesca Troilo ◽

Francesca Malagrinò ◽

Lorenzo Visconti ◽

Angelo Toto ◽

Stefano Gianni

Keyword(s):

Protein Interactions ◽

Specific Interaction ◽

Sh2 Domain ◽

Regulatory Subunit ◽

Wild Type ◽

Protein Protein Interactions ◽

Sh2 Domains ◽

Trans Isomerization ◽

A Site ◽

Kinetics Of

SH2 domains are protein domains that modulate protein–protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the βE and βF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

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Identifying protein–protein interactions in somatic hypermutation

Journal of Experimental Medicine ◽

10.1084/jem.20050161 ◽

2005 ◽

Vol 201 (4) ◽

pp. 493-496 ◽

Cited By ~ 3

Author(s):

Myron F. Goodman ◽

Matthew D. Scharff

Keyword(s):

Mouse Model ◽

Protein Interactions ◽

Somatic Hypermutation ◽

Model Systems ◽

Antigen Binding ◽

Immunoglobulin Genes ◽

Protein Protein Interactions ◽

Cell Systems ◽

High Affinity ◽

Key Players

Somatic hypermutation (SHM) in immunoglobulin genes is required for high affinity antibody–antigen binding. Cultured cell systems, mouse model systems, and human genetic deficiencies have been the key players in identifying likely SHM pathways, whereas “pure” biochemical approaches have been far less prominent, but change appears imminent. Here we comment on how, when, and why biochemistry is likely to emerge from the shadows and into the spotlight to elucidate how the somatic mutation of antibody variable (V) regions is generated.

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Following evolutionary paths to protein-protein interactions with high affinity and selectivity

Nature Structural & Molecular Biology ◽

10.1038/nsmb.1670 ◽

2009 ◽

Vol 16 (10) ◽

pp. 1049-1055 ◽

Cited By ~ 54

Author(s):

Kalia Bernath Levin ◽

Orly Dym ◽

Shira Albeck ◽

Shlomo Magdassi ◽

Anthony H Keeble ◽

...

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

High Affinity ◽

Evolutionary Paths

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Study of two types of protein-protein interactions : i, a high affinity antigen-antibody complex and ii, a transient complex between an oncoprotein and a methyltransferase

10.32657/10356/74114 ◽

2018 ◽

Author(s):

◽

Anagha Tambe

Keyword(s):

Protein Interactions ◽

Protein Protein Interactions ◽

High Affinity ◽

Transient Complex ◽

Antibody Complex ◽

Antigen Antibody

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Functional Display of Bioactive Peptides on the vGFP Scaffold.

10.21203/rs.3.rs-257370/v1 ◽

2021 ◽

Author(s):

Sharon Min Qi Chee ◽

Jantana Wongsantichon ◽

Sze Yi Lau ◽

Barindra Sana ◽

Yuri Frosi ◽

...

Keyword(s):

Protein Interactions ◽

Bioactive Peptides ◽

Negative Regulator ◽

Live Cells ◽

Structural Studies ◽

Protein Protein Interactions ◽

Target Interaction ◽

High Affinity ◽

Novel Scaffold

Abstract Grafting bioactive peptides into recipient protein scaffolds can often increase their activities by conferring enhanced stability and cellular longevity. Here, we describe use of vGFP as a novel scaffold to display peptides. vGFP comprises GFP fused to a bound high affinity Enhancer nanobody that potentiates its fluorescence. We show that peptides inserted into the linker region between GFP and the Enhancer are correctly displayed for on-target interaction, both in vitro and in live cells by pull-down, measurement of target inhibition and imaging analyses. This is further confirmed by structural studies highlighting the optimal display of a vGFP-displayed peptide bound to Mdm2, the key negative regulator of p53 that is often overexpressed in cancer. We also demonstrate a potential biosensing application of the vGFP scaffold by showing target-dependent modulation of intrinsic fluorescence. vGFP is relatively thermostable, well-expressed and inherently fluorescent. These properties make it a useful scaffold to add to the existing tool box for displaying peptides that can disrupt clinically relevant protein-protein interactions.

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