Two Structures of Cyclophilin 40 Paul Taylor, Jacqueline Dornan, Amerigo Carrello, Rodney F Minchin, Thomas Ratajczak, Malcolm D Walkinshaw  Structure  Volume 9, Issue 5, Pages 431-438 (May 2001) DOI: 10.1016/S0969-2126(01)00603-7

Figure 1 Alignment of Selected Cyclophilin Proteins Sequence alignment of human cyclophilin A (CYPH_HUMAN), bovine and human cyclophilin 40 (CYP4_BOVIN and CYP4_HUMAN), WIS2 from S. Pombe (CYP4_SCHPO), and CPR6 and CPR7 from S. cerevisiae (CPR6_YEAST and CPR7_YEAST). The sequences were aligned using Clustal W [29]. In bold are the features discussed in the text, including conserved cysteines and glutamates and residues forming the inserted loop. The 13 residues important in defining the active site of the cyclophilin family of proteins are shown starred above the text. The high degree of conservation between these cyclophilin domains (residues 1–183) is clearly seen Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 3 Stereo Pictures of the Monoclinic and the Tetragonal Folding Intermediate Forms of Bovine Cyclophilin 40 (a) The stereo picture of the monoclinic form of bovine cyclophilin 40 was drawn using Molscript [30]. The cyclophilin domain (residues 1–183) is colored green. Side chains of selected active site residues (Arg75, Phe80, Phe133, His141, His146) are colored black. Glu96, which forms hydrogen bonds to the divergent loop, is also shown. The backbone of the linker (residues 184–213) joining the cyclophilin and TPR domains is shown in black. Selected salt bridges between the positively charged residues of helix Q (K244, K245, K248, R251) with linker residues D204 and D200 (D204/OD…K248/N = 2.99 Å, D200/OD2… K245/N = 2.73 Å, D200/OD1…K248/N = 3.16 Å) are shown as dashed green lines. Side chains of all charged residues on the TPR domain are shown. Arg and Lys, blue; Glu and Asp, red. There are seven helices in the TPR domain: P (20 residues, V216-K235), Q (21 residues, W239-A259), R (24 residues, D262-K285), S (12 residues, W289-L300), T (13 residues, T307-G319), U (14 residues, Y323-I336), and V (18 residues, K341-K362). The slight distortion in helix R is caused by the presence of Pro270. The portions of the helices corresponding to the repeating TPR motifs shown in Figure 2 are colored dark blue (TPR1, helices P and Q, residues 223–256; TPR2, helices R and S, residues 273–306; TPR3, helices T and U, residues 307–340). The protruding C-terminal portion of helix V is the putative calmodulin domain and is colored magenta. (b) The stereo picture of the tetragonal folding intermediate form of bovine cyclophilin 40 was drawn using Molscript [30]. Coloring is the same as (a). Helices T, U, and V are not visible in the electron density. Helices are colored according to the fully folded monoclinic form in (a): Helix P (20 residues, V216-K235), Q (21 residues, W239-A259), R (24 residues, D262-K285), S (12 residues, W289-D298). The portions of the helices corresponding to the repeating TPR motifs shown in Figure 2 are colored dark blue (TPR1, residues 223–256; TPR2, residues 273–298). Alternative conformations of residues Ala260 and Ser287 are primarily responsible for helices R and S adopting an extended rod-like helical conformation Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 3 Stereo Pictures of the Monoclinic and the Tetragonal Folding Intermediate Forms of Bovine Cyclophilin 40 (a) The stereo picture of the monoclinic form of bovine cyclophilin 40 was drawn using Molscript [30]. The cyclophilin domain (residues 1–183) is colored green. Side chains of selected active site residues (Arg75, Phe80, Phe133, His141, His146) are colored black. Glu96, which forms hydrogen bonds to the divergent loop, is also shown. The backbone of the linker (residues 184–213) joining the cyclophilin and TPR domains is shown in black. Selected salt bridges between the positively charged residues of helix Q (K244, K245, K248, R251) with linker residues D204 and D200 (D204/OD…K248/N = 2.99 Å, D200/OD2… K245/N = 2.73 Å, D200/OD1…K248/N = 3.16 Å) are shown as dashed green lines. Side chains of all charged residues on the TPR domain are shown. Arg and Lys, blue; Glu and Asp, red. There are seven helices in the TPR domain: P (20 residues, V216-K235), Q (21 residues, W239-A259), R (24 residues, D262-K285), S (12 residues, W289-L300), T (13 residues, T307-G319), U (14 residues, Y323-I336), and V (18 residues, K341-K362). The slight distortion in helix R is caused by the presence of Pro270. The portions of the helices corresponding to the repeating TPR motifs shown in Figure 2 are colored dark blue (TPR1, helices P and Q, residues 223–256; TPR2, helices R and S, residues 273–306; TPR3, helices T and U, residues 307–340). The protruding C-terminal portion of helix V is the putative calmodulin domain and is colored magenta. (b) The stereo picture of the tetragonal folding intermediate form of bovine cyclophilin 40 was drawn using Molscript [30]. Coloring is the same as (a). Helices T, U, and V are not visible in the electron density. Helices are colored according to the fully folded monoclinic form in (a): Helix P (20 residues, V216-K235), Q (21 residues, W239-A259), R (24 residues, D262-K285), S (12 residues, W289-D298). The portions of the helices corresponding to the repeating TPR motifs shown in Figure 2 are colored dark blue (TPR1, residues 223–256; TPR2, residues 273–298). Alternative conformations of residues Ala260 and Ser287 are primarily responsible for helices R and S adopting an extended rod-like helical conformation Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 2 Alignment of TPR Sequences for Hsp90 Binding Proteins Proteins that contain three copies only of the TPR motif and that have been shown to interact with Hsp90 were identified from the literature and are given in the table, using Swissprot names. Additionally, the TPR2 domain of Hop has been included. Each TPR motif from the above proteins was manually aligned. The proteins were bovine and human cyclophilin 40 (CYP4_BOVIN, CYP4_HUMAN); human FKBP 51 and 52 (FKBP5_HUMAN and FKBP4_HUMAN); human protein phosphatase 5 (PPP5_HUMAN); Cpr 6, Cpr 7, and Cns1 from S. cerevisiae (CYP6_YEAST, CYP7_YEAST, and CNS1_YEAST); and human Hop (IEFS_HUMAN). Numbering above the sequences runs from 1 to 34 and gives the position of a particular amino acid relative to the start of the TPR motif. The horizontal cylinders below each alignment designate the extent of helices P, Q, R, S, T, and U in the 34 amino acid sequence, using structural data from Cyp40 to assign helices. A broken cylinder indicates the helix begins or terminates earlier or later in the protein sequence, therefore failing to conform to the classical, idealized helix (13 aa) turn (3 aa) helix (14 aa) model for TPR motifs. Arrows projecting from the cylinders point to the positions in the sequence where the helices start and finish. The three linker residues between helix AI and helix BI are underlined. Closed and shaded cylinders illustrate a complete helix within the TPR motif. Only TPR 3 conforms to the idealized structure. Residues identified as being important in binding Hsp90 are bold and underlined. Structurally conserved residues are italicized and bold Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 4 Stereo Cα Trace of the Monoclinic Form of Bovine Cyclophilin Amino Acids Are Labeled Every 20th Residue Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 5 Stereo Picture of the Main Intermolecular Interaction in the Tetragonal Form of Cyp40 and an Overlay of the Folding Intermediate Dimer and the Fully Folded Conformer (a) The partially unfolded TPR domains form a symmetrical dimer. One molecule is colored as in Figure 3b, and the partner molecule is colored red. The first TPR, comprising helices P and Q (shown in the boxed region), make an intermolecular contact with helix R′ of the dimer-related molecule. (b) The boxed region of (b), with the same color scheme, showing helices P, Q, and R′ for the tetragonal form, overlaid with helices P, Q, and R (yellow) of the folded TPR domain of the monoclinic form. The intra- and intermolecular interactions of helix R with helices P and Q are seen to be nearly identical Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 5 Stereo Picture of the Main Intermolecular Interaction in the Tetragonal Form of Cyp40 and an Overlay of the Folding Intermediate Dimer and the Fully Folded Conformer (a) The partially unfolded TPR domains form a symmetrical dimer. One molecule is colored as in Figure 3b, and the partner molecule is colored red. The first TPR, comprising helices P and Q (shown in the boxed region), make an intermolecular contact with helix R′ of the dimer-related molecule. (b) The boxed region of (b), with the same color scheme, showing helices P, Q, and R′ for the tetragonal form, overlaid with helices P, Q, and R (yellow) of the folded TPR domain of the monoclinic form. The intra- and intermolecular interactions of helix R with helices P and Q are seen to be nearly identical Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)

Figure 6 Stereo Picture of the Main Intermolecular Interaction in the Monoclinic Form of Cyp40 The yellow and blue molecules are related by a unit cell translation along b of 47.3 Å. The C-terminal residues EKAAY from one molecule (blue) fit into a groove of an adjacent molecule (yellow). The groove is formed by 2 residues from each of the three TPR helices (A1,2,3): Lys227, Phe234, Ser274, Asn278, Lys308, and Arg312 (side chains shown). Tyr365 fits into a hydrophobic pocket in the groove and is the last residue visible in the electron density. (The C-terminal 5 residues are presumably structurally disordered in the crystal.) Structure 2001 9, 431-438DOI: (10.1016/S0969-2126(01)00603-7)