Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).
Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047, United States.
Natalie A
NA
Ciaccio
T Steele
TS
Reynolds
C Russell
CR
Middaugh
Jennifer S
JS
Laurence
D051696:Activating Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D052160:Benzothiazoles; D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D016350:Leucine Zippers; D009154:Mutation / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013844:Thiazoles / Q000737:chemistry; D014633:Valine / Q000737:chemistry*
C507532:ATF5 protein, human; D051696:Activating Transcription Factors; D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D014633:Valine