Introduction
Peptidyl prolyl
cis-trans
isomerase (PPIase) (EC5.2.1.8) catalyzes
cis
–
trans
isomerization of the Xaa– Pro peptide bonds of proteins [1]. Because the peptidebond in the
cis
conformation is energetically unfavor-able compared with that in the
trans
conformation[2,3], and
cis
–
trans
isomerization of this peptide bondis intrinsically very slow [4], prolyl isomerization isregarded as a rate-limiting step of the folding reactionof proteins that contain
cis
prolines in a folded state[5]. PPIases are divided into four structurally unrelatedfamilies: FK506-binding proteins (FKBPs), cyclophi-lines, parvulins and the Ser
⁄
Thr phosphatase 2Aactivator, PTPA [6].FKBP22 is a member of the FKBP family and pres-ent in various Gram-negative bacteria [7–9]. It is ahomodimer, in which each subunit consists of anN-terminal domain (N-domain) and a C-terminalPPIase domain (C-domain). Based on its similarities tothe macrophage-infectivity potentiator (MIP) proteinfrom
Legionella pneumophila
in amino acid sequence,FKBP22 has been classified as a member of the MIP-like FKBP subfamily [7].
Escherichia coli
FkpA is alsoa member of this subfamily [10]. Of the members of this subfamily,
L. pneumophila
MIP [11] and
E. coli
FkpA [12] are the only ones for which the crystalstructures have been determined. These structuresstrongly resemble each other, having an rmsd of 0.8 A ˚for all C
a
atoms. According to these structures, theseproteins assume a nonglobular V-shaped homodimericstructure, in which two monomers interact with eachother at their N-domains. Each monomer assumes adumbbell-like structure, in which the N-domain (con-sisting of
a
1 and
a
2 helices) and the C-domain [con-sisting of six
b
strands (
b
1–
b
6) and an
a
4 helix] arelinked by a 40-residue-long
a
3 helix. As a result, theC-domains, which are located at both ends of aV-shaped structure, face each other across the cleft of this structure. The interface of two monomers, whichis located at the bottom of the V-shaped structure, isstabilized by the hydrophobic interactions between the
a
1 helix of one monomer and the
a
2 helix of the other.However, the role of a V-shaped structure of MIP-likeFKBP subfamily proteins remains to be understood.We have previously shown that FKBP22 from thepsychrotrophic bacterium
Shewanella
sp. SIB1 existsas a homodimer and exhibits PPIase activity for bothpeptide and protein substrates [8]. SIB1 FKBP22shows amino acid sequence identities of 56% to
E. coli
FKBP22 [7], of 43% to
E. coli
FkpA [10] andof 41% to
L. pneumophila
MIP [13]. Construction of the mutant proteins N-domain
+
and C-domain
+
,which lack the C- and N-domains of SIB1 FKBP22,respectively, followed by biochemical characterizationof these proteins, suggest that the C-domain isrequired for PPIase activity, and the N-domain isrequired for dimerization and efficient binding to aprotein substrate of PPIase [14,15]. However, itremains to be determined whether a V-shaped struc-ture is required for efficient binding of SIB1 FKBP22to a protein substrate, because the N-domain
+
, whichretains the ability to bind to a protein substrate, con-tains the entire
a
3 helix and therefore may be able toassume a V-shaped homodimeric structure. Attemptsto construct the N-domain without the
a
3 helix haveso far been unsuccessful because of the instability of the protein.In this report, we constructed the mutant protein,NNC-FKBP22, in which the N-terminal region (Met8– Ala60) is repeated twice within a single polypeptidechain, and characterized it biochemically. This mutantprotein was designed such that the repetitive N-termi-nal region is folded into a structure similar to that of the
Shewanella
sp. SIB1 FKBP22 wild-type protein(WT), which has a homodimeric structure. Based onthese results, we discuss the role of a V-shaped struc-ture of FKBP22.
Results
Design of monomeric mutant protein
The monomeric mutant protein (NNC-FKBP22) wasdesigned based on a model of the 3D structure of WT(SIB1 FKBP22) (Fig. 1A), which has previously beenreported [14]. According to this model, WT assumes aV-shaped homodimeric structure, like those of
L. pneu-mophila
MIP [11] and
E. coli
FkpA [12]. In this struc-ture, the Ala60 of one monomer is located in closeproximity to the Met8 of the other monomer. Bothresidues are located close to the bottom of the cleft of the V-shaped structure. Therefore, it is stronglyexpected that the mutant protein, termed NNC-FKBP22, in which Met1–Ala60 of SIB1 FKBP22 isattached to Met8–Ile205 of SIB1 FKBP22 throughthree glycine residues, is monomeric and folded into astructure similar to that of WT without one arm of the‘V’. A model of the 3D structure of NNC-FKBP22 isshown in Fig. 1A. Its primary structure is also sche-matically shown in Fig. 1B in comparison with that of WT. NNC-FKBP22 and SIB1 FKBP22 (WT) in a His-tagged form will be designated as NNC-FKBP22 andSIB1 FKBP22 (WT), respectively, hereafter.
Engineering of monomeric FKBP22
C. Budiman
et al.
4092
FEBS Journal
276
(2009) 4091–4101
ª
2009 The Authors Journal compilation
ª
2009 FEBS
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