Anfinsen Comes out of the Cage During Assembly of the Bacterial Pilus

Commentary

Anfinsen comes out of the cage during assembly of the bacterial pilus

Staffan Normark*

Microbiology and Tumorbiology Center, Karolinska Institute, 171 77 Stockholm, Sweden

he classic experiments of Anfinsen information to their substrate proteins. by providing steric information in the Tand his associates established that the DnaK is thought to bind to unfolded form of the missing strand. The subunit primary sequence of a polypeptide con- proteins via exposed hydrophobic resi- groove occupied by the G1 strand also tains all of the information needed for a dues, preventing their aggregation and participates in subunit–subunit interac- polypeptide to fold into its native fully misfolding, until they are released into tions in the pilus. Thus, donor strand active tertiary structure (1). Although An- the cytoplasm, where folding is com- complemention couples the folding of finsen’s conclusions still hold, for the most pleted (6, 7). The substrates for DnaK the subunit with the capping of its inter- part, it is now becoming clear that certain can be either newly synthesized proteins active groove, ensuring that the groove is proteins, such as the subunit proteins or proteins misfolded because of stress never free to interact nonproductively in building up adhesive surface organelles on conditions. the periplasm. During pilus assembly, Escherichia coli, need additional steric in- In contrast to this cytoplasmic general which occurs at the outer membrane formation from other proteins in order to folding machinery described above, re- usher, the N-terminal extension of an fold properly (2). In their paper published cent work suggests that the small incoming subunit displaces the chaper- in this issue of PNAS, Barnhart et al. (2) periplasmic PapD-like chaperones, one G1 strand and occupies the groove of show that at least some proteins may which participate in the assembly of ad- the most recently incorporated subunit require information for folding to be tran- hesive surface structures in many Gram- (donor strand exchange) (Fig. 1B). The siently provided by distinct molecular negative pathogens, facilitate folding by mature pilus thus consists of an array of chaperones. directly providing steric information to subunits, each of which contributes a There are many chaperones involved their substrates. PapD was the first chap- strand to complete the fold of its neigh- in protein folding; however, few are erone for which the crystal structure was bor. Pili are heteropolymeric structures known to act by directly donating steric solved (8). It contains two immunoglob- containing a number of different subunit information to their substrate proteins. ulin-like (Ig) domains oriented in a boo- proteins assembled in a specific order. For example, the GroEL-GroES chaper- merang shape. PapD specifically inter- The affinity of the incoming donor one system couples ATP hydrolysis to acts with pilus subunits before their strand for the exposed hydrophobic cleft the iterative binding and release of un- incorporation into P pilus polymers in in the preceding subunit may in part folded polypeptides. The binding stabi- uropathogenic strains of E. coli (9, 10). determine the order of subunit incorpo- lizes the substrate in an unfolded state The protein was characterized as a chap- ration. Pilus assembly by PapD-like until it is released and allowed to fold. erone because it is required for pilus chaperones thus represents a distinct The GroEL (3) chaperone is a multi- formation but does not appear in the variation of the Anfinsen principle, be- meric ATP-driven macromolecular com- pilus itself. PapD is one chaperone in a cause subunits require information from plex that has been proposed to facilitate family of over 30 members that are in- another polypeptide entity—the chaper- protein folding via the Anfinsen cage volved in the assembly of adhesive struc- one and then the neighboring sub- model (4, 5). This model is based on the tures on the bacterial cell surface. Two unit—to attain and maintain, respec- view that protein folding in vivo is limited recently published crystal structures of tively, their proper fold. In these regards, by intermolecular reactions that produce PapD-like chaperones in complex with PapD-like chaperones function similarly aggregation. It proposes that the GroEL pilus subunits, or pilins, reveal the basis to intramolecular chaperones (IMCs), cavity provides a sequestered microen- for their interaction with subunits (11, covalently attached peptides that have vironment in which folding to the native 12). Pilus subunits lack the C-terminal been shown to facilitate protein folding ␤ state can proceed while the substrate is seventh -strands that would otherwise by providing steric information and that protected from aggregation. Proteins complete their Ig folds. The missing are subsequently cleaved from their sub- that require the GroEL chaperonin for in strand results in a groove along the sur- strates (13, 14). However, unlike IMCs, vivo folding do not receive steric infor- face of the pilin that exposes its hydro- PapD-like chaperones are separate, fully mation from this class of chaperones. phobic core. In the structures, the chap- folded proteins that provide steric infor- ␤ Rather, these chaperones act to prevent erone donates a -strand (strand G1) mation for pilin folding while simul- or overcome the misfolding of their sub- that occupies the groove and completes taneously capping their interactive strate polypeptides. The DnaK (Hsp70) the fold of the subunit (donor strand surfaces. family represents another major class of complementation) (Fig. 1A). In the ab- The Hultgren group has now proceeded cytoplasmic chaperones (6, 7). In con- sence of the chaperone, subunits are to test whether the provision of the miss- junction with the co-factors DnaJ and unstable and are degraded by proteases GrpE, DnaK couples ATP hydrolysis such as DegP, which recognizes dena- with substrate binding and release. Sim- tured, misfolded, and͞or aggregated pro- See companion article on page 7709. ilar to the GroEL system, the DnaK teins. Donor strand complementation *To whom reprint requests should be addressed. E-mail: family members do not contribute steric thus allows the subunit to correctly fold [email protected].

7670–7672 ͉ PNAS ͉ July 5, 2000 ͉ vol. 97 ͉ no. 14 Downloaded by guest on September 30, 2021 strand) that allows pilin subunits to fold properly. Donor strand complementation and exchange also suggest a molecular model for another general role of chaperones in organelle biogenesis. PapD caps subunit interactive surfaces and prevents inap- propriate interactions until the subunit has reached its proper assembly site. This is strikingly reminiscent of the invariant chain, which occupies the MHC-class-II peptide-binding groove during folding to prevent nonproductive peptide binding (17). In both of these cases, the interactive groove is first transiently occupied by a chaperone to prevent premature interactions. Subsequently, the chaperone is exchanged for the final substrate, which now permanently occupies the groove. The many chaperones that participate in organelle biogenesis in bacte- ria and eukaryotic cells may act in an analagous fashion. The findings presented by the Hult- gren group have important practical im- plications for vaccine development. Fig. 1. Pilin domain topology diagrams. Dashes indicate additional polypeptide not shown. (A) In donor FimH is a promising vaccine candidate strand complementation, the chaperone contributes its G1 strand (red) to complete the immunoglobulin- against lower urinary tract infections like fold of the subunit (white). The completed fold is noncanonical because the G1 strand runs parallel to (16). However, purification of the intact the subunit C-terminal F strand. The N-terminal extension is shown as a blue strand. (B) After donor strand exchange, the N-terminal extension of one subunit completes the Ig fold of its neighbor in a canonical adhesin directly from the polymerized manner, as the N-terminal extension runs anti-parallel to the F strand. (C) Donor-strand-complemented fiber is extremely inefficient and requires FimH (dscFimH) was constructed by fusing the N-terminal extension of FimG (blue), which is predicted to the presence of detergents. In addition, complete the fold of FimH in the pilus, to the C terminus of FimH with a 4-amino-acid linker (yellow). The FimH cannot be purified when expressed topology of the receptor-binding domain is not shown, but its position relative to the dscFimH pilin alone because it is proteolytically de- domain is indicated by the labeled box. graded. Thus, pilus-associated adhesins have been prepared from the periplasmic COMMENTARY ing strand to pilus subunits obviates the and thus allow it to fold in the absence of space in complex with their cognate requirement for the chaperone during pi- the chaperone. Indeed, unlike wild-type chaperones and have been used as pro- lin folding (2). They make use of the FimH, dscFimH can be expressed as a tective antigens (16). By adding the miss- mannose-binding FimH lectin assembled proteolytically stable protein with wild- ing strand to pilus-associated adhesins, it into the tip structure of type 1 pili. These type mannose-binding activity in the bac- should now be possible to produce pro- pili are expressed by most E. coli strains terial periplasm in the absence of the teolytically stable adhesins in large quan- and have been shown to be required for E. periplasmic chaperone FimC. Also in in tities in the absence of chaperone. There coli to cause bladder infections in mice as vitro folding assays, denatured dscFimH, is good hope that such structurally ‘‘re- well as in primates (15, 16). The FimH unlike denatured wild-type FimH, was paired’’ adhesins could also act as pro- lectin consists of an N-terminal lectin do- capable of resuming its native ␤-sheet tective antigens because the receptor main and a C-terminal pilin domain. They CD-structure. As predicted by the donor binding region of known adhesins are have constructed a FimH variant, donor- strand exchange model, the dscFimH pro- located in a domain that is apart from the strand-complemented FimH (dscFimH), tein was not incorporated into pili in the domain that requires additional steric which has been extended at its C terminus presence of chaperone and usher because information. by a sequence corresponding to the N the groove of the fully folded dscFimH is In summary, the biogenesis of disease- terminus of FimG, which is thought to presumably occupied by the donor strand associated bacterial pili has generated fas- complete the Ig fold of the pilin domain of and thus not free either to interact with cinating novel insights regarding chaper- FimH in the mature pilus (Fig. 1C). The the chaperone or to undergo subsequent one-assisted protein folding through do- donor strand complementation and ex- donor strand exchange. The results indi- nor strand complementation and also has change model suggests that this donor cate that the mechanism of action of a provided an explanation for ordered pro- strand should occupy the groove and com- small chaperone like PapD is to provide tein incorporation into heteropolymeric plete the fold of the pilin domain of FimH the missing information (i.e., the missing structures.

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