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Stabilizing IkappaBalpha by "consensus" design.

ABSTRACT: IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advantage of a native Trp residue in ankyrin repeat (AR) 6 and engineered Trp residues in AR2, AR4 and AR5, we show that the cooperative transition involves AR2 and AR3, while the non-cooperative transition involves AR5 and AR6. The major structural transition can be affected by single amino acid substitutions converging to the "consensus" ankyrin repeat sequence, increasing the native state stability significantly. We further characterized the structural and dynamic properties of the native state ensemble of IkappaBalpha and the stabilized mutants by H/(2)H exchange mass spectrometry and NMR. The solution experiments were complemented with molecular dynamics simulations to elucidate the microscopic origins of the stabilizing effect of the consensus substitutions, which can be traced to the fast conformational dynamics of the folded ensemble.

SUBMITTER: Ferreiro DU

PROVIDER: S-EPMC1866275 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Stabilizing IkappaBalpha by "consensus" design.

Ferreiro Diego U DU Cervantes Carla F CF Truhlar Stephanie M E SM Cho Samuel S SS Wolynes Peter G PG Komives Elizabeth A EA

Journal of molecular biology 20061115 4

IkappaBalpha is the major regulator of transcription factor NF-kappaB function. The ankyrin repeat region of IkappaBalpha mediates specific interactions with NF-kappaB dimers, but ankyrin repeats 1, 5 and 6 display a highly dynamic character when not in complex with NF-kappaB. Using chemical denaturation, we show here that IkappaBalpha displays two folding transitions: a non-cooperative conversion under weak perturbation, and a major cooperative folding phase upon stronger insult. Taking advanta ...[more]

PMID: 17174335

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Project description:Many pathogenic viruses, including influenza virus, Ebola virus, coronaviruses, and Pneumoviruses, rely on class I fusion proteins to fuse viral and cellular membranes. To drive the fusion process, class I fusion proteins undergo an irreversible conformational change from a metastable prefusion state to an energetically more favorable and stable postfusion state. An increasing amount of evidence exists highlighting that antibodies targeting the prefusion conformation are the most potent. However, many mutations have to be evaluated before identifying prefusion-stabilizing substitutions. We therefore established a computational design protocol that stabilizes the prefusion state while destabilizing the postfusion conformation. As a proof of concept, we applied this principle to the fusion protein of the RSV, hMPV, and SARS-CoV-2 viruses. For each protein, we tested less than a handful of designs to identify stable versions. Solved structures of designed proteins from the three different viruses evidenced the atomic accuracy of our approach. Furthermore, the immunological response of the RSV F design compared to a current clinical candidate in a mouse model. While the parallel design of two conformations allows identifying and selectively modifying energetically less optimized positions for one conformation, our protocol also reveals diverse molecular strategies for stabilization. We recaptured many approaches previously introduced manually for the stabilization of viral surface proteins, such as cavity-filling, optimization of polar interactions, as well as postfusion-disruptive strategies. Using our approach, it is possible to focus on the most impacting mutations and potentially preserve the immunogen as closely as possible to its native version. The latter is important as sequence re-design can cause perturbations to B and T cell epitopes. Given the clinical significance of viruses using class I fusion proteins, our algorithm can substantially contribute to vaccine development by reducing the time and resources needed to optimize these immunogens.

Dataset Information

Stabilizing IkappaBalpha by "consensus" design.

Publications

Stabilizing IkappaBalpha by "consensus" design.

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