Towards conformational fidelity of a quaternary HIV-1 epitope: computational design and directed evolution of a minimal V1V2 antigen

Enzymes are proteins that catalyse chemical reactions and, as such, have been widely used to facilitate a variety of natural and industrial processes, dating back to ancient times. In fact,...

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Artificial Enzymes Made to Order: Combination of Computational Design and Directed Evolution

Angewandte Chemie International Edition ◽

10.1002/anie.200802865 ◽

2008 ◽

Vol 47 (41) ◽

pp. 7802-7803 ◽

Cited By ~ 19

Author(s):

Thomas R. Ward

Keyword(s):

Directed Evolution ◽

Computational Design ◽

Artificial Enzymes

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Toward New Nonnatural TIM-Barrel Enzymes Using Computational Design and Directed Evolution Approaches

Understanding Enzymes ◽

10.1201/b19951-20 ◽

2016 ◽

pp. 561-611

Author(s):

Mirja Krause ◽

Rik Wierenga

Keyword(s):

Directed Evolution ◽

Computational Design ◽

Tim Barrel

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Computational design of novel fullerene analogues as potential HIV-1 PR inhibitors: Analysis of the binding interactions between fullerene inhibitors and HIV-1 PR residues using 3D QSAR, molecular docking and molecular dynamics simulations

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2008.10.039 ◽

2008 ◽

Vol 16 (23) ◽

pp. 9957-9974 ◽

Cited By ~ 64

Author(s):

Serdar Durdagi ◽

Thomas Mavromoustakos ◽

Nikos Chronakis ◽

Manthos G. Papadopoulos

Keyword(s):

Molecular Dynamics ◽

Molecular Docking ◽

Molecular Dynamics Simulations ◽

Computational Design ◽

3D Qsar ◽

Binding Interactions ◽

Dynamics Simulations ◽

Hiv 1

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A substrate selected by phage display exhibits enhanced side-chain hydrogen bonding to HIV-1 protease

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318006691 ◽

2018 ◽

Vol 74 (7) ◽

pp. 690-694 ◽

Cited By ~ 1

Author(s):

Ian W. Windsor ◽

Ronald T. Raines

Keyword(s):

Crystal Structure ◽

Hydrogen Bonding ◽

Molecular Recognition ◽

Hydrogen Bonds ◽

Phage Display ◽

Directed Evolution ◽

Side Chain ◽

Endogenous Substrates ◽

Hiv 1 ◽

Additional Hydrogen

Crystal structures of inactive variants of HIV-1 protease bound to peptides have revealed how the enzyme recognizes its endogenous substrates. The best of the known substrates is, however, a nonnatural substrate that was identified by directed evolution. The crystal structure of the complex between this substrate and the D25N variant of the protease is reported at a resolution of 1.1 Å. The structure has several unprecedented features, especially the formation of additional hydrogen bonds between the enzyme and the substrate. This work expands the understanding of molecular recognition by HIV-1 protease and informs the design of new substrates and inhibitors.

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Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518405113 ◽

2016 ◽

Vol 113 (16) ◽

pp. 4446-4451 ◽

Cited By ~ 13

Author(s):

Jordan R. Willis ◽

Jessica A. Finn ◽

Bryan Briney ◽

Gopal Sapparapu ◽

Vidisha Singh ◽

...

Keyword(s):

Amino Acid ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Computational Design ◽

Cell Repertoire ◽

B Cell Repertoire ◽

Amino Acid Length ◽

Massive Scale ◽

Amino Acid Mutations ◽

Hiv 1

Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1–naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1–naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 107unique HCDR3 amino acid regions from 70 different HIV-1–naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1–naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts.

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Directed Evolution of Retrovirus Envelope Protein Cytoplasmic Tails Guided by Functional Incorporation into Lentivirus Particles

Journal of Virology ◽

10.1128/jvi.79.2.834-840.2005 ◽

2005 ◽

Vol 79 (2) ◽

pp. 834-840 ◽

Cited By ~ 15

Author(s):

Christoph A. Merten ◽

Jörn Stitz ◽

Gundula Braun ◽

Eric M. Poeschla ◽

Klaus Cichutek ◽

...

Keyword(s):

Directed Evolution ◽

Cleavage Site ◽

Leukemia Virus ◽

Cytoplasmic Tail ◽

Hiv Protease ◽

Evolution System ◽

Peptide Cleavage ◽

C Terminus ◽

Hiv 1 ◽

Vector Particles

ABSTRACT In contrast to most gammaretrovirus envelope proteins (Env), the Gibbon ape leukemia virus (GaLV) Env protein does not mediate the infectivity of human immunodeficiency virus type 1 (HIV-1) particles. We made use of this observation to set up a directed evolution system by creating a library of GaLV Env variants diversified at three critical amino acids, all located around the R-peptide cleavage site within the cytoplasmic tail. This library was screened for variants that were able to functionally pseudotype HIV-1 vector particles. All selected Env variants mediated the infectivity of HIV-1 vector particles and encoded novel cytoplasmic tail motifs. They were efficiently incorporated into HIV particles, and the R peptide was processed by the HIV protease. Interestingly, in some of the selected variants, the R-peptide cleavage site had shifted closer to the C terminus. These data demonstrate a valuable approach for the engineering of chimeric viruses and vector particles.

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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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