Amino acid requirements for formation of the TGF-beta-latent TGF-beta binding protein complexes.
Transforming growth factor beta (TGF-beta) is secreted primarily as a latent complex consisting of the TGF-beta homodimer, the TGF-beta propeptides (called the latency-associated protein or LAP) and the latent TGF-beta binding protein (LTBP). Mature TGF-beta remains associated with LAP by non-covalent interactions that block TGF-beta from binding to its receptor. Complex formation between LAP and LTBP is mediated by an intramolecular disulfide exchange between the third 8-cysteine (8-Cys3) domain of LTBP with a pair of cysteine residues in LAP. Only the third 8-Cys domains of LTBP-1, -3, and -4 bind LAP. From comparison of the 8-Cys3(LTBP-1) structure with that of the non-TGF-beta-binding 8-Cys6(fibrillin-1), we observed that a two-residue insertion in 8-Cys3(LTBP-1) increased the potential for disulfide exchange of the 2-6 disulfide bond. We further proposed that five negatively charged amino acid residues surrounding this bond mediate initial protein-protein association. To validate this hypothesis, we monitored binding by fluorescence resonance energy transfer (FRET) analysis and co-expression assays with TGF-beta1 LAP (LAP-1) and wild-type and mutant 8-Cys3 domains. FRET experiments demonstrated ionic interactions between LAP-1 and 8-Cys3. Mutation of the five amino acid residues revealed that efficient complex formation is most dependent on two of these residues. Although 8-Cys3(LTBP-1) binds proTGF-betas effectively, the domain from LTBP-4 does so poorly. We speculated that this difference was due to the substitution of three acidic residues by alanine, serine, and arginine in the LTBP-4 sequence. Additional experiments with 8-Cys3(LTBP-4) indicated that enhanced binding of LAP to 8-Cys3(LTBP-4) is achieved if the residues A, S, and R are changed to those in 8-Cys3(LTBP1) (D, D, and E) and the QQ dipeptide insertion of LTBP-4 is changed to the FP in 8-Cys3(LTBP-1). These studies identify surface residues that contribute to the interactions of 8-Cys3 and LAP-1 and may yield information germane to the interaction of 8-Cys domains and additional TGF-beta superfamily propeptides, an emerging paradigm for growth factor regulation.
Chen
Yan
Y
Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA.
Ali
Tariq
T
Todorovic
Vesna
V
O'leary
Joanne M
JM
Kristina Downing
A
A
Rifkin
Daniel B
DB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053779:Latent TGF-beta Binding Proteins; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D048008:TNF Receptor-Associated Factor 3 / Q000737:chemistry / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; C509032:LTBP1 protein, human; C509040:LTBP4 protein, human; D053779:Latent TGF-beta Binding Proteins; D046911:Macromolecular Substances; D020033:Protein Isoforms; D048008:TNF Receptor-Associated Factor 3; D016212:Transforming Growth Factor beta; D003545:Cysteine