Specific Recognition of the Collagen Triple Helix by Chaperone HSP47

Abstract

this finding, we examined the relationship between the structure of Yaa ؊3 and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa ؊3 determined the binding affinity for HSP47. Maximal binding was observed when Yaa ؊3 was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr ؊3 and one Arg 0 . The results revealed that HSP47 recognizes the Yaa ؊3 and Arg 0 residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47. Heat-shock protein 47 (HSP47)3 is an essential molecular chaperone for normal procollagen biosynthesis in mammals. Disruption of both alleles of the hsp47 gene in mice causes abnormal procollagen folding in the endoplasmic reticulum (ER), resulting in an embryonic lethal phenotype (1, 2). Although the molecular function of HSP47 remains unclear, it has been suggested that HSP47 could stabilize correctly folded triple-helical intermediates of procollagen that are otherwise unstable at body temperature (3, 4). HSP47 has also been suggested to inhibit lateral associations that form insoluble aggregates in the ER (5). Efforts toward elucidating the collagen-recognition mechanism of HSP47 have been made by our group and others, and to date, Arg residues at Yaa positions in the collagenous Gly-Xaa-Yaa repeats have been shown to be necessary for interaction with HSP47 (6, 7). The importance of the triple-helical structure for HSP47 binding has also been strongly suggested, and it was finally proven in the preceding paper by using conformationally constrained collagenous peptides (8). The minimal number of Arg residues per triple helix required for the specific interaction was further determined to be one. In addition, the Yaa residue occupying position Ϫ3 (denoted as Yaa Ϫ3 , and similar nomenclatures are used later), based on the essential Arg residue at a Yaa position (whose position is defined as position 0), has also been suggested to be important for the interaction, since replacement of the 4-hydoxy-Lproline (Hyp) residue at position Ϫ3 in (Pro-Hyp-Gly) 2 -Pro-Hyp Ϫ3 -Gly-Pro-Arg 0 -Gly-(Pro-Hyp-Gly) 4 -Pro-Hyp-amide with p-benzoyl-Lphenylalanine (Bpa) abolished the binding, even though the peptide had a triple-helical structure and contained an Arg residue at a Yaa position.In the present study, we focused on the structure of Yaa Ϫ3 , which was expected to be another structural determinant for HSP47 binding, together with the side chain structure of Arg 0 . A structure-activity relationship study was performed using coll...

Procollagen Triple Helix Assembly: An Unconventional Chaperone-Assisted Folding Paradigm

“…(2). In our opinion, however, the reported data for HSP47 binding to triple helical peptides [17], [19], [40] and to collagen [15], [35] do not support the cooperative binding model proposed in [15].…”

“…Note that this is not inconsistent with the well-documented ability of HSP47 to bind to gelatin [35], [38] since a significant fraction of gelatin chains may be folded into triple helices [39]. Thus, we decided to evaluate the extent of the triple helix stabilization expected for type I procollagen based on the binding constants reported in [40].…”

“…(5),(6). After substitution of the dissociation constants reported in [40] and T 0 ≈307 K measured above into Eqs. (5),(6), we calculated T 0 + δT as a function of HSP47 concentration without any adjustable parameters.…”

“…(5),(6) based on the HSP47 binding sites and dissociation constants reported in [40], as described in Methods (Theoretical analysis). Two possible values of the dissociation constant K d (OGxRG) = 4.6 µM (solid line) and K d (OGxRG) = 0.94 µM (dashed line) were used to account for the uncertainty in HSP47 binding at OGxRG, where x is a variable amino acid.…”

“…(5),(6) based on the HSP47 dissociation constants K i f and K j u for yGxRG sequences reported in [40], where y and x indicate variable residues. Since K j u ≥1 mM [40], we neglected the corresponding terms in Eq. (5).…”