Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti–HIV-1 drugs

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Abstract
During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. Significance The envelope subunit gp41 is an attractive target for therapeutic intervention against HIV-1. Interfering with the interaction between the heptad-repeat regions of gp41 is a promising approach to inhibit HIV-1 fusion to the host cell membrane. Here, we present an alternative rational design and protein-engineering approach to produce highly stable single-chain proteins that accurately mimic the trimeric coiled-coil surface of the gp41 N-terminal heptad repeat. This approach has a strong potential for development to HIV-1 drugs, vaccines, or microbicides and could be extendable to the design of proteins interfering with other types of coiled-coil interactions.
Funding Information
  • European Commission (EC) (HEALTH-F3-2007-201038)
  • Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness) (BIO2012-39922-C02-01/02)
  • Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness) (BIO2008-00750-E)
  • Consejería Economía, Innovación, Ciencia y Empleo, Junta de Andalucia (Ministry of Economy, Innovation, Science and Employment, Government of Andalucia) (P09-CVI-5063)
  • Consejería Economía, Innovación, Ciencia y Empleo, Junta de Andalucia (Ministry of Economy, Innovation, Science and Employment, Government of Andalucia) (Incentivos-BOJA-5-1-2008)

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