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Refined structure of rat Clara cell 17 kDa protein at 3·0 Å resolution
J. Mol. Biol. (1992) 224, 441-448
Refined Structure of Rat Clara Cell 17 kDa Protein at 3-OA Resolution Timothy C. Urnland’?, S. Swaminathan’S, William Furey’, Gurmukh Singh’ James Pletcher’ and Martin Sax’ ‘Biocrystallography Laboratory, VA Medical Center PO Box 12055, University Dr C, Pittsburgh, PA 15240, U.S.A. and University
of
2VA Medical Center and Department of Pathology Pittsburgh School of Medicine, Pittsburgh, PA 15240, U.S.A.
(Received 3 June 1991; accepted 26 November 1991) The rat Clara cell 17 kDa protein (previously referred to as the rat Clara cell 10 kDa protein) has been reported to inhibit phospholipase A, and papain, and to also bind progesterone. It has been isolated from rat lung lavage fluid and crystallized in the space group P6,22. The structure has been determined to 3-OA resolution using the molecular replacement method. Uteroglobin, whose amino acid sequence is 55.7% identical, was used as the search model. The structure was then refined using restrained least-squares and simulated annealing methods. The R-factor is 22.5%. The protein is a covalently bound dimer. Two disulfide bonds join the monomers together in an antiparallel manner such that the dimer encloses a large internal hydrophobic cavity. The hydrophobic cavity is large enough to serve as the progesterone binding site, but access to the cavity is limited. Each monomer is composed of four a-helices. The main-chain structure of the Clara cell protein closely resembles that of uteroglobin, but the nature of many of the exposed side-chains differ. This is true, particularly in a hypervariable region between residues 23 and 36, and in the HlH4 pocket. Keywords:
Clara cell 17 kDa protein; Clara cell 10 kDa protein; uteroglobin;
amniotic fluid from rat and human, being secreted from the fetal lung (Singh et al., 1986). The rat Clara cell 17 kDa protein (RLL§) has three isotypes in the p1 range 4.6 to 50. It has been suggested that the isotypes arise from co- or post-translational modifications of a single peptide (Singh et al., 1990). RLL isotypes are covalently bonded dimers made up of two monomers of 77 residues each (Katyal et al., 1990). The monomers are linked together by two disulfide bonds in an antiparallel manner. The molecular weight of the dimer, determined from its amino acid sequence, is 16,908 daltons. Prior to the availability of sequence information, the molecular weight of the dimer was estimated to be - 10 kDa from sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Singh et al., 19853). For this reason, the protein has been referred to in the literature as
1. Introduction Clara cells are a non-ciliated, non-mucous type of secretory cell (Kuhn et al., 1974) which predominate in the distal airways. These cells have been reported to be involved in the renewal of damaged distal airway epithelium and in the metabolism of xenobiotics (Evens et al., 1978; Boyd, 1977). The Clara cell 17 kDa protein was first detected during a search for cell-specific markers for use in the study of airway cells in normal and pathological states (Singh & Katyal, 1984). The 17 kDa protein has been identified thus far in the lung lavage fluid from, and is specific to, secretory granules of Clara cells of all species examined: rat, mouse, hamster, dog, monkey and human (Singh et al., 1985a; Bedetti et al., 1987). It has also been observed in the t In partial fulfillment of the Doctor Degree. $ Author to whom all correspondence addressed.
of Philosophy should
$ Abbreviations used: RLL, Clara cell 17 kDa from rat lung lavage fluid; PLA,, phospholipase uteroglobin.
be
441 0022-2836/92/060441-08
$03.00/O
progesterone
0
1992 Academic
protein A,; UG,
Press Limited
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T. C. Urnland
et al.
Table 1 Amino
acid sequences of RLL 5
UG RLL
and rabbit
UG
10
15
~GICPRFAHVIENLLLGTPS SSDICPGFLQVLEALLLGSES
UC RLL
20 25 SYETSLKEFEPDDTMKDAG NYEAALKPFNPASDLQNAG
UG RLL
40 M Q M K TQLKRLVDTLPQ
UG
K
RLL
KLTEKILTSPLCEQDLRV
30
K
V
45 L
K
I
V
60
L
A common numbering One-letker amino acid Glycine; H, histidine; I, glutamine; R; arginine;
T
D
35
S
L
P
50 Q
S
P
L
C
65
E
K
T T R ETRINIV
55 N
I
~~ --
~
E
70
M
M
75
-
~-
scheme is used, based on the sequence homology. code: A, alanine; C, cysteine; D, aspartate; E, glutamate; F, phenylalanine; G, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P; proline; Q, S, serine; T, threonine; V; valine; W, tryptophan; Y, tyrosine.
the Clara cell 10 kDa protein. It has been observed that RLL is an inhibitor of pancreatic phospholipase A, (PLAJ and papain, and binds progesterone (Singh et al., 1990). However, it is unknown what, if any, physiological relevance this may have. Comparison of the amino acid sequences of RLL and of rabbit uteroglobin (UG) revealed that 39 of the 70 residues (557%) contained in the UG monomer were identical to the corresponding residues in the RLL monomer (Katyal et al., 1990). The amino acid sequences of RLL and UG are given in Table 1. UG is a protein contained in the uterine washings of pregnant rabbit (Krishnan et al., 1967; Beier, 1968) and two related species, hare and pica (Nieto & Lombardero, 1982). UG has also been observed in airway cells and in the male genital tract, as well as an UG-like protein in the circulation of rabbit (Warembourg et al., 1986; Beier et al., 1975; Kikukawa & Mukherjee, 1989). It is reported to have immunosuppressive, anti-inflammatory, progesterone binding, and PLA, and protease inhibiting properties (Manjunath et al., 1984; Miele et al., 1988; Urzua et al., 1970; Levin et al., 1986; Gupta et al., 1987). It has been suggested that UG plays a role in control of inflammation and platelet aggregation, and in the regulation of cellular signal transduction (Mukherjee et al., 1988; Kikukawa & Mukherjee, 1989). Like RLL, UG is also a dimer but with 70 amino acid residues per monomer (Ponstingl et al., 1978; Popp et al., 1978), and also with two disulfide bonds between monomers. Six different crystal forms of UG have been grown (Mornon et al., 1978, 1979, 1980; Buehner & Beato, 1978), with the C222, crystal structure being solved to 1.34 A resolution (1 b = @l nm; Morize et al., 1987). Despite the amino acid sequence similarity, UG and RLL have been found to be antigenically distinct (Singh et al., 1990) as well as possess different tissue distributions. A hypervariable
region in the sequences between UG and RLL has been noted at residues 23 to 36 (Singh et al.; 1988). (The common numbering system for the sequences of RLL and UG proposed by Singh et al. (1990) is being used.) The possibly important function of RLL in the lung, together with further elucidation of its differences and similarities with UG, made a structural study . of interest. The homogeneity suggested the use of the molecular replacement method (rotation and translation functions), using UG as the model, to determine the crystal structure of RLL.
2. Materials
and Methods
(a) Crystallization RLL crystals exhibit polymorphism, dependent on f&e at which they were grown. Thus far, cubic (Swaminathan et al., 1990), monoclinic and hexagonal forms have been observed. The hexagonal crystals were grown using the sitting drop vapor diffusion method. A solution composed of 40 ~1 of 100 ,ug protein/ml (isotype III) and 5 ~1 of 60 y0 (w/v) saturated ammonium sulfate in 10 mlvr-Tris . HCl buffer (pH 7.1) was equilibrated against a reservoir solution of 60% saturated ammonium sulfate in 10 mm-Tris HCl buffer (pH 7.1). Small needle-shaped crystals grew to their maximum size (-05 mm x @05 mm x 005 mm) within a week. These crystals have space group P6522, with cell dimensions a = b = 5207 A and c = 109.27 a. Assuming a monomer weight of $454 daltons and one monomer per asymmetric unit, the Matthews (1968) coefficient was calculated to be 2.53 A”! dalton, which is in the accepted range. In this case, more than 1 monomer/asymmetric unit leads to unacceptably low Matthews coefficients (less than 127 A’/dalton): and thus the calculation is definitive. pB
(b) Data collection
and processing
X-ray diffraction data for the native crystals were collected using a Siemens multi-wire area detector. A
Structure
of Rat Clara
Table 2 Observed rejections
as a function
of resolution
%
d (4
Total
Observed
(in range)
% (cumulative)
co-46 46-39 3.9-34 34-3.0
613 358 453 602
603 346 393 381
98.4 967 868 63.3
984 97.7 94.2 850
A reflection was considered observed if I > 20(l).
Rigaku RU200 rotating anode operated at 42 kV and 65 mA with a focal spot of @2 mm x 20 mm was used to produce Ni-filtered CuK, radiation. The X-ray beam was focused using Franks mirrors. Data collection used the software described by Blum et al. (1987). Data were processed using the XENGEN package (Howard et al., 1987) and locally modified scaling programs of Weissman (1982). Although the crystals suffered substantial decay and diffracted X-rays only weakly, due to their small size, data collected from a single crystal to 3.7 A resolution were obtainable and used in the molecular replacement solution. These data were later merged with data from 4 other crystals to obtain a 3.0 A data set. The merged data set was reduced to 1733 unique reflections with I2 20(I). At 3.0 A resolution the data are 85% complete (Table Z), with a merging R-factor: of 11.7% for the 5 crystals. (c) Molecular replacement The structure of the hexagonal crystal form of RLL was solved using the molecular replacement method. Only the 3.7 A data set collected from a single crystal was used in these calculations. First, the self-rotation function (Rossmann & Blow, 1962) was calculated using the Crowther fast rotation function as implemented in ROTRAN (Crowther, 1972; Craven, 1975). Data between 50 A and 40.0 A resolution and a Patterson radius of 12.0 A were used. This only revealed the presence of 622 symmetry, with no significant peaks due to additional non-crystallographic symmetry being observed. Thus, the RLL dimer’s 2-fold axis must coincide with a crystallographic 2-fold axis, consistent with the presence of 1 monomer/asymmetric unit. The orientation of the RLL monomer in the hexagonal cell was then determined using MERLOT (Fitzgerald, 1988). Specifically, Crowther’s fast rotation function was used for a coarse search and Lattman’s rotation function (Lattman 8: Love, 1972) was used for a fine search of Eulerian space. The search model was the rabbit UG monomer from its 6222, crystal form at 1.34 A resolution (Morize et al., 1987). In this structure the UG dimer also made use of a crystallographic 2-fold axis. Cross-rotation functions were calculated using 2 different models: (1) all the atoms of the UG monomer were included; and (2) only main-chain and Cfi atoms of the UG monomer were included (the poly-alanine model). The rotation functions were calculated using 1> 3a(l) data. For the Crowther rotation function, resolution ranges of 8.0 A or 7.0 A to 3.7 A and a Patterson radius of 20 A were used. For the Lattman rotation function, resolution ranges of 8.0 A or 7.0 A to 3.9 A were ‘used. The highest peak in all of the rotation functions calculated, using both models, consistently was at or near the Eulerian angles a = 30.0”,
Cell 17 LDa Protein
443
fi = 81.0” and y = 1800” (as defined by MERLOT). The UG search models’ original orientations were such that their dimer 2-fold axes were parallel to the RLL hexagonal cell’s b-axis at the Euler angles a = /l = y = 0”. Thus, this solution of the rotation function indicates that the UG search models had been orientated in the RLL cell such that their dimer 2-fold axes lay parallel to the crystallographic 2-fold axis in the [- 1 1 0] direction. This result is consistent with that of the self-rotation function. The translation problem of the molecular replacement method was solved using an R-factor minimum search, where:
R = W’ol -JW’,II/W’~I> calculated by RMAP (W. Furey, unpublished results). The R-factor search was also used at the same time to determine the correct space group. The Laue symmetry was known to be 6/mmm, but due to the limited number of observed 001 reflections and the enantiomorphic nature of the space group possibilities, the space group could not be determined unambiguously by the systematic absences. Thus, the R-factor search had to be conducted in each of the 6 possible space groups. The fact that the dimer’s 2-fold axis coincides with a crystallographic 2-fold axis restricted the dimer’s center of mass to lie on a given line residing on specific planes for each possible space group. From the orientation of the search model in the RLL cell given by the rotation function, the R-factor minimum search was reduced to a search over the set of fractional co-ordinates of {z, --2, z}, where z = 90, 5/12, 516, l/4, l/6 and l/12 for the space group P622, P6,22, P6,22, P6,22, P6,22 and P6,22, respectively. Prior to the R-factor minimum search, the UG dimer co-ordinates were translated so that its center of mass was at the origin, rotated to the optimum orientation as determined from the rotation function, and then translated along z to the value appropriate for each search. The search model was then reduced to only one monomer containing only main-chain and CB atoms, which was translated along the line (2, --2, z}. The minimum value of R ( =5@8%) was obtained in space group P6,22, with the poly-alanine UG monomer positioned such that the center of mass of the dimer was at x = 0.575, y = -0575 and z = l/12. The minimum R-factors obtained for the other 5 possible space groups ranged between 57.5 ye and 68.8%. The results of the R-factor searches are given in Table 3. All observed reflections from the 3.7 a data set were used. This initial orientation and position of the UG polyalanine monomer model in the P6,22 RLL cell was then
Table 3 Minimum R-factors calculated for the orientated UG poly-alanine monomer search model in each of the possible space groups Space group P622 P6,22 P6,22 P6,22 P6,22 P6,22
Min. Et at: 06882 05865 0.5750 06544 05940 0.5077
z
Y
z
0.500 0575 0575 0450 0%50 0.575
- 0500 - 0.575 -0.575 -0.475 - 0.856 - 0.575
PO, l/2 5/12, 11/12 5/6, l/3 l/4, 3/4 l/6, 2/3 l/12, 7/12
The fractional co-ordinates give the position of the dimer’s center of mass at which the minimum R-factpr is obtained. The 2 different values of z for each space group correspond to shifts of the origin by l/2 unit cell along the c-axis.
t R = UFol -W’cll/Wol.
T. 6. Umtand
444
refined by least-squares using the rigid-body refinement program GREF (Furey, 1990). For the first 11 cycles, the model was treated as a single rigid body. Then for cycles I2 through 17 the model was broken up into 4 rigid bodies, with each roughly corresponding to one of the 4 cc-helices contained in the UG monomer. The 4 rigid bodies were UG residues 1 to 15; 16 to 29, 30 to 48 and 49 to 70. This gave R = 45.1 To for 1046 reflections to 3.7 d resolution, with I > 3@). (d) Model building
and rejhement
The refined co-ordinates of the poly-alanine UG search model were used to calculate the initial phases of a 2F0 - F, map, with the 1085 observed reflections to 3.7 d having FO> 2a(FO). Density corresponding to that expected for the large side-chains and disulfide bonds of RLL was observed. This confirmed the correctness of the solution, as only CB side-chain atoms had been included in the phase calculations. The RLL sequence was then built into the 2F,-F, map using the FRODO/TOM software (Jones, 1982; Cambillau & Horjales, 1987) on a Silicon Graphics Personal Iris computer. This first RLL model led to an R = 41.4%. Refinement was then begun using GPRLSA (Furey et al., 1982), which is a modified version of Hendrickson’s restrained least-squares refinement package PROLSQ (Hendrickson & Konnert, 1980). Initially, only the 3.7 A data set was used, but later data from all 5 crystals were merged into a single data set and included in the refinement. Cycles of refinement were alternated with manual corrections of the model using 2FO-- Fc and F,- F, maps, and occasionally residuedeleted 2F,- F, maps. In the later stages of refinement, a modified version of GPRLSA was used that restrained the 2 disulfide bonds that exist between monomers. This modification was necessary since GPRLSA will not normally restrain bonds that cross an asymmetric unit boundary. When the R-factor had reached 3@8% for 3.0 a data, refinement was continued using XPLOR (Bruenger et al., 1987). After the use of XPLOR, the modified version of GPRLSA was used again to restore the disulfide bonds to reasonable geometry, since XPLOR is also incapable of dealing correctly with disulfide bonds spanning asymmetric units. The final R-factor was 22.5o/o for 1661 observed reflections in the resolution range
et al. !@O A to 3.0 8, and the deviations from ideal geometry were acceptable (Table 4). Due to the lack of high resolution data, only an overall thermal factor was used, where B3= 7.23 8’. Eo solvent molecules, except for a singie sulfate ion, have been placed in the model. The electron density, which has been modeled by a sulfate ion, lies between the side-chain amino groups of Lys62 and the 2-fold symmet’ry-related residue in an adjoining dimer. In the final 2F,-F, map, the only residues for which the main-chain was not well-defined were Srg74 and Val75. ,4s a check that the final RLL model was not overly biased by the initial UG model, residue-deleted 2F0--F, maps were calculated. Specifically, a 5 residue segment in the RLL model was deleted from the phase calculation if t,he side-chain of the central residue in the corresponding segment in UG had a greatly different size or shape. The maps were then viewed using FRODO/TOM, and density for all of the deleted residues was as expected for the RI& sequence. Also, the RLL chain extends further on both the h’ and C-terminal ends than does UG, and residuedeleted maps confirmed the positions of these residues. The RLL refined monomer co-ordinates and structure factors have been deposited at the Brookhaven Protein Data Bank, with the reference codes 1CCD and RICCDSF.
3. Results and Discussion The native P6,22 form of RLL is a covaientl?; bound dimer that has exact (crystallographic) 2-fold symmetry between its monomers (Fig. 1). Two disulfide bonds, Cys3 to Cys69’ and Cys69 to CysJ’, join the monomers together in an antiparallel manner. (Residues contained in the second monomer of the dimer are indicated with a prime.) Only two hydrogen bonds act to stabilize the dimer. These are between Gln40 and Thr52’ and the symmetry-relat,ed pair. It would appear that in addition to the disulfide bonds, hydrophobic forces are important in maintaining the dimer. The
Table 4 Deviations from ideal geometry restrained refinement
after
0
r.m.s. deviation
A. Distance restraints (A)
Bond distance Angle Planar Planar Chiral
distance l-4 distance restraint volume (A3)
B. Non-bonded contacts (A) Single torsion Multiple torsion Possible H-bond C. Torsion angles (deg.) Planar Staggered ( + / - 60, 180”) Orthonormal ( + / - 90”) r.m.s., root-mean-square.
0.020 0.045 0.035 0.020 0.200
0.016 0.062 0040 0.009 0.284
0300 0300 0300
0216 0.248 0174
50 150 150
2.6 26.3 322
Figure 1. A ribbon cartoon (Priestle, 1988) of the RLL dlmer viewed down the crystallographic 2-fold axis. The 2 disulfide bonds between the monomers are indicated by bold continuous lines.
Structure of Rat Clara Cell 17 kDa Protein
Figure 2. The C” skeleton of the RLL monomer. The residue labels follow the common numbering scheme for RLL and UG (Singh et al., 1990).
monomer is composed of four a-helices, as shown in Figure 2. However, the monomer does not exhibit the four a-helical bundle motif (Weber & Salemme, 1980) as the helices of the two monomers composing the dimer interact to create a large internal cavity. Helix 1 encompasses Pro4 through Leu14. A /?-turn (Leul3 to Serl7) leads into helix 2 (Glu18 to Lys26). Following the second helix is another b-turn consisting of Lys26 to Asn29. Helix 3 extends between Ser32 and Thr47, and helix 4 ranges from Gln50 to Leu64. No /?-structure is present. The chain ends have no particular secondary structure. The five proline residues present in the monomer tend to break the structure into different segments. The main-chain dihedral angles for the helical regions had mean values of (4) = - 61.0” and (9) = -41.4”. All main-chain dihedral angles are in allowed regions of conformational space. Asn29 lies in the left-handed helical region of the Ramachandran map (Ramachandran et al., 1963).
445
The corresponding residue in UG, Glu29, also has this conformation. The root-mean-square deviation between main-chain atoms of residues 1 to 70 of RLL and UG is @SO8. A large hydrophobic cavity exists between the two monomers of the dimer. The residues that define this cavity are Phe6, Va19, LeulO, Leul3, Leu14, Tyr21, Leu25, Gly38, Leu41, Va145, Ile56, Leu59, Thr60, Ile63 and the symmetry-related residues on the second monomer. The distances between residues lining the cavity and the corresponding residues related by 2-fold symmetry give an indication of the width of the cavity. For example, the distance between Thr60 Cyl and Thr60’ CY1 is 1@18, and between Leu25 Cy and Leu25’ Cy is 23.6 8. The length of the cavity along the 2-fold axis may be approximated by the distance between Phe6 CE2and Va145’ Cy2’, which is 13.5 8. However, it appears that the cavity is not highly accessible to the outside environment (Fig. 3). This cavity is very similar to that of UG, both in geometry and in residue make-up. Of the 14 residues per monomer that line the cavity, 11 are identical between UG and RLL. Conservative substitutions have occurred for the three residues which differ. It has been suggested that the large internal cavity contained within UG is the binding site for progesterone based on its hydrophobic nature and geometry (Mornon et al., 1980; Morize et al., 1987). By analogy, the cavity within RLL may be suspected of being the progesterone binding site as well. However, these sites are controversial due to limited access to the cavities. In addition, only the reduced form of either protein binds progesterone, while the native proteins are in the oxidized form that exhibits little or no binding. No oxidized UG-hormone complex could be isolated from the uterus (Tancredi et al., 1982). Nevertheless, model building demonstrated that the dimensions of the interior cavity of RLL would allow a progesterone molecule to reside there, analogous to the proposed UG-progesterone model, if several side-chains moved slightly from their positions in the native
Figure 3. A stereo view of the RLL dimer with all atoms included. The view is normal to the 2-fold axis. Notice that there is limited access to the interior cavity.
446
T. C. Urnland et al.
Figure 4. Stereo view of the RLL interior cavity with a molecule of progesterone modeled inside. The view is down the dimer’s Z-fold axis. Only the amino acid residues that line the cavit,y are displayed (continuous lines), except Phe6 and PheS’, which have been omitted for clarity. The progesterone (broken lines) has been fitted according to the proposed UG-progesterone model.
structure (Pig. 4). The closest contact is bet,ween Cd1 of Leul3 and Cl9 of the hormone, at 1.8 A. Side-chains of LeulS’, Leu41 and Leu41’ contact the hormone at distances of 2.0 to 2.5 A, and those of LeulO, LeulO’ and Ile56 are 2.5 to 3.0 A away. The only close contact involving a main-chain atom of les$ than 4.0 A is between the carbonyl 0 of Ile56 and C21 of progesterone, at 3.2 A. Hydrogen bonds may exist between Oq of Tyr21 and TyRl’ of RLL and 03 and 020 of progesterone, as in the UG-progesterone model. Atomic co-ordinates for progesterone were taken from Campsteyn et al. (1972). No electron density was observed which could be attributed to bound progesterone in the hydrophobic cavity or at any other site in the RLL crystal structure. However, since no progesterone was added to the mother liquor, this absence of electron density only demonstrates that the RLL was isolated and crystallized in the unbound state; and does not disprove the internal cavity binding site hypothesis. If the cavity is the binding site, then lack of access to the cavity in the oxidized RLL structure would correlate with its non-binding of the hormone. The reduction of the protein’s disulfide bonds may allow the two monomers to partially separate, and thus form an access channel into the cavity for the hormone. Clearly the structure of the protein-hormone complex needs to be determined to provide added insight regarding the binding site.
ends of the dimer form a’ The C and N-terminal pocket. This is separated from the large internal cavity by Phe6 and Phe6’, but’ a rotation of the phenyl groups would open a small channel into the internal cavity. This pocket is deeper in RLL than the corresponding pocket in UG, due to the additional residues contained at both ends of RLL. RLL possesses surface pockets that are formed by helix 1 and helix 2 (HlH2) and by helix 1 and helix 4 (HlH4), as does UG. HlH2 is formed by Gly5, Gln8, Va,l9, Ala24, Lys26, Pro27 and Phe28. HlH4 is formed by Aspl, Ile2, Leu7? Gln8, LeulO, Glull, Va157, Thr60, Glu61, Leu64 and Thr65. Although the main-chain conformation of these pockets is similar for both UG and RLL, the residue compositions are different. For HlH2, only two of the seven residues that form the pocket are ident,ical in both proteins. Likewise, for HlH4, only four of the 11 residues in the pocket are identical. The HlH2 pocket of RLL has one basic and no acidic residues, whereas UG has three basic and one acidic residue. The HlH4 pocket of RLL has three acidic and no basic residues, and UG has two basic and two acidic residues in the same pocket. Thus? the pockets of the two proteins differ in both charge and hydropathy. Also, the HlH4 pocket of RLL is smaller than that of UG. This is due to the substitution iu RLL of a leucine for an alanine at residue seven, and a different main-chain geometry near the N
Figure 5. A stereo view of the superimposed amino acid residues from RLL and UG that form the HlH4 pocket. For RLL (continuous lines) these residues are Aspl, Ile2, Leu7, Glu8, LenlO, Glul I, Va157, Thr60, GluGI, Leu64 and Thr65. For UG (broken lines) these residues are Glyl, Ile2, Ala7, Hi&, IlelO, Glul I. Met57, Thr60, Glu61, Va164 and Lys65. The RLL pocket is smaller mainly due to the substitution of leucine for alanine at residue 7.
Structure
of Rat Clara
terminus causing the side-chain of Ile2 of RLL to project further out into the pocket than the corresponding side-chain in UG (Fig. 5). Preliminary nuclear magnetic resonance studies have indicated that interaction between progesterone and oxidized UG in solution caused conformational changes localized to the HlH4 pocket region (Jamin et al., 1989). If this conformation change is relevant to hormone binding, then perhaps the decreased ability of RLL to bind progesterone as compared to UG (Singh et al., 1990) is due to the differing properties of the HlH4 pockets in the two proteins. Singh et al. (1990) reported the presence of a hypervariable region in the RLL and UG amino acid sequences between residues 23 and 36, where only 4 of the 14 residues are identical. However, the main-chain conformation in this region is similar for both proteins, containing the carboxyl end of helix 2, the amino end of helix 3, and the turn connecting the helices. Again, RLL and UG differ in the number of charged side-chains each contains in this region. RLL has one basic and one acidic residue, and UG has two basic and five acidic residues in this section. Much of this region is exposed to solvent and so it may give rise to the functional differences
between the two proteins. We thank Dr J. P. Mornon for kindly providing us with the atomic co-ordinates of UG. This work was supported by the U.S. Army Medical Research and Development Command under Intragovernmental order no. 87PP7852. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. An Andrew Mellon Predoctoral Fellowship sponsored by the A. W. Mellon Educational and Charitable Trust and administered by the University of Pittsburgh was held by T.C.U. References Bedetti, C. D.; Singh, J., Singh, G.; Katyal, S. L. & Wong-Chong, M. L. (1987). Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoelonal antibodies. J. Histochem. Cytochem. 35, 789-794. Beier, H. M. (1968). Uteroglobin: a hormone-sensitive endometrial protein involved in blastocyst development. Biochim. Biophys. Acta, 160, 2899291. Beier, H. M., Bohn, H. & Muller, W. (1975). Uteroglobin-like antigen in the male genital tract secretions. Cell Tiss. Res. 165, l-11. Blum, M., Metcalf; P., Harrison, S. C. 85 Wiley, D. C. (1987). A system for collection and on-line integra’tion of X-ray diffraction data from a multiwire area detector. J. Appl. Crystallogr. 20, 235-242. Boyd, M. R. (1977). Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung. Nature (London), 269, 713-715. Bruenger, A. T., Kuriyan, J. & Karplus, M. (1987). Crystallographic R factor refinement by molecular dynamics. Science, 235, 4588460. Buehner, M. & Beato, M. (1978). Crystallization and preliminary crystallographic data of rabbit uteroglobin. J. Mol. Biol. 120, 337-341. Cambillau, C. & Horjales, E. (1987). TOM: a FRODO subpackage. J. Mol. Graph. 5, 174.
Cell 17 IcDa Protein
447
Campsteyn, H., DuPont, L. & Dideberg, 0. (1972). Structure cristalline et moleculaire de la progesterone CzlH,,O,. Acta Crystallogr. sect. B, 28, 3032-3042. Craven, B. M. (1975). Computer programs ROTRAN for the determination of crystal structures which contain rigid body fragments of known structure. Univ. of Pittsburgh, Dept. of Crystallography, Technical Report TR-75-2. Crowther, R. A. (1972). The fast rotation function. In The Molecular Replacement Method (Rossmann, M. G., ed.), pp. 173-178, Gordon and Breach, New York. Evens, M. J., Cabral-Anderson, L. J. & Freeman, G. (1978). Role of Clara cell in renewal of the bronchiolar epithelium. Lab. Invest. 38, 648-655. Fitzgerald, P. M. D. (1988). MERLOT, an integrated package of computer programs for the determination of crystal structures by molecular replacement. J. Appl. Crystallogr. 21, 273-278. Furey, W. (1990). PHASES-a program package for the processing and analysis of diffraction data from macromolecules. American Crystallographic Association Fortieth Anniversary Meeting, New Orleans, LA, abstract PA33. Furey, W., Wang, B. C. & Sax, M. (1982). Crystallographic computing on an array processor. J. Appl. Crystallogr. 15, 160-166. Gupta, R. P., Patton, S. E.: Jetten, A. M. & Hook, G. E. R. (1987). Purification, characterization and proteinase-inhibitory activity of a Clara cell secretory protein from the pulmonary extracellular lining of rabbits. Biochem. J. 248, 337-344. Hendrickson, W. A. & Konnert, J. H. (1980). Stereochemically restrained crystallographic leastsquares refinement of macromolecule structures. In Biomolecular Structure, Conformation, Function and Evolution (Srinivasan, R., ed.), vol. 1, pp. 43-57, Pergamon Press, Oxford. Howard, A. J., Gilliland, G. L., Finzel, B. C. & Poulos, T. L. (1987). The use of an imaging proportional counter in macromolecular crystallography. J. Appl. Crystallogr. 20, 383-387. Jamin, N., Roy, P., Fridlansky, F., Delepierre, M., Milgrom, E., Roques, B. P. & Mornon. J. P. (1989). Preliminary assignments of the aromatic and some methyl group resonances of the ‘H-NMR spectrum of bhe oxidized form of uteroglobin. Eur. J. Biochem. 183, 219-226. Jones, T. A. (1982). FRODO: a graphics fitting program for macromolecules. Computational In Crystallography (Sayre, D., ed.), pp. 3033317, Clarendon Press, Oxford. Katyal, S. L., Singh, G., Brown, W. E., Kennedy, A. L., Squeglia, N. & Wong-Chong, M. L. (1990). Clara cell secretory (10 k daltons) protein: amino acid and oDNA sequences, and developmental expression. Prog. Respir. Res. 25, 29-35. Kikukawa, T. & Mukherjee, A. B. (1989). Detection of a uteroglobin-like phospholipase A, inhibitory protein in the circulation of rabbits. Mol. Cell. Endocrinol. 62, 177-187. Krishnan, R. S. & Daniel, J. C. Jr (1967). “Blastokinin”: inducer and regulator of blastocyst development in the rabbit uterus. Science, 158, 490-492. Kuhn, C. III, Callaway, L. A. & Askin, F. B. (1974). The formation of granules in the bronchiolar Clara cells of the rat. J. Ultrastruct. Res. 49, 387-400. Lattman, E. E. $ Love, W. E. (1972). A rotational search procedure for detecting a known molecule in a crystal. Acta Crystallogr. sect. B, 26, 1854-1857.
448
T. C. Urnland
Levin, S. W., Butler, J. DeB., Schumacher, U. K., Wightman, P. D. & Mukherjee; A. B. (1986). Uteroglobin inhibits phospholipase A, activity. Life sci. 38, 1813-1819. Manjunath, R., Chung, S. I. & Mukherjee, A. B. (1984). Crosslinking of uteroglobin by transglutaminase. Biochem. Biophys. Res. Commun. 121, 400-407. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Miele, L., Cordella-Miele, E., Facchiano, A. & Mukherjee, A. B. (1988). Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin 335, 726-730. and lipocortin I. Nature (London), Morize, I., Surcouf, E.; Vaney, M. C., Epelboin, Y., Buehner, M., Fridlansky, F., Milgrom, E. & Mornon, J. P. (1987). Refinement of the C222, crystal form of oxidized uteroglobin at 1.34A resolution. J. Mol. Biol. 194, 725-739. Mornon, J. P., Surcouf, E., Bally, R., Fridlansky, F. & Milgrom, E. (1978). X-ray analysis of a progesteronebinding protein (uteroglobin): preliminary results. J. Mol.
Biol.
122, 237-239.
Mornon, J. P., Bally, R., Fridlansky, F. & Milgrom, E. (1979). Characterization of two new crystal forms of uteroglobin. J. Mol. Biol. 127, 237-239. Mornon, J. P., Fridlansky, F., Bally, R. & Milgrom, E. (1980). X-ray crystallographic analysis of a progesterone binding protein. The 6222, crystal form of oxidized uteroglobin at 2.2 A resolution. J. Mol. Biol. 137, 415-429. Mukherjee, A. B.; Cordella-Miele, E., Kikukawa, T. & Miele, L. (1988). Modulation of cellular response to antigens by uteroglobin and transglutaminase. In Advances in Exp. Med. and Bio.: Advances in Post-translational Modifications of Proteins and Aging
(Zappia, V., Galletti, P., Porta, R. 85 Wold, F., eds), vol. 231, pp. 135152, Plenum Press, New York. Nieto, A. & Lombardero, M. (1982). Uteroglobin-like antigens in species of Lagomorpha. Comp. Biochem. PhysioE. B, 71, 511-514. Ponstingl, H., Nieto, A. & Beato, M. (1978). Amino acid sequence of progesterone-induced rabbit uteroglobin. Biochemistry, 17, 390883912. Popp, R. A., Foresman, K. R., Wise, L. D. & Daniel, J. C., Jr (1978). Amino acid sequence of a progesterone-binding protein. Proc. Nat. Acad. Sci., U.S.A. 75, 5516-5519. Priestle, J. P. (1988). RIBBON: a stereo cartoon drawing program for proteins. J. Appl. Crystallogr. 21, 572-576. Ramachandran, G. N., Ramakrishnan, C. & Sasisekharan, V. (1963). Stereochemistry of polypeptide chain configurations. J. Mol. Biol. 7, 95-99.
Edited
et al.
Rossmann, M. G. & Blow, D. M. (1962). The detection of subunits within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24-31. Singh, G. & Katyal, S. L. (1984). An immunologic study of the secretory products of rat Clara cell. J. Histochem.
Cytochem.
32, 49-54.
Singh, G., Katyal, S. L.; Ward, J. M., Gott,ron, S. A., Wong-Chong, M. L. & Riley, E. J. (1985a). Secretory proteins of the lung in rodents. J. Histochem. Cytochem.
33, 564-568.
Singh, G., Katyal, S. L. & Gottron, S. A. (19853). Antigenic molecular and functional heterogeneity of Clara cell secretory proteins in rat. Biockim. Biophys. Acta, 829, 156-163. Singh, G., Katyal, S. L. & Wong-Chong, M. L. (1986). A quantitative assay for a Clara cell-specific protein and its application in the study of development of pulmonary airways in the rat. Pediatr. Res. 20, 802-805. Singh, G., Katyal, S. L., Brown, W. E., Phillips, S., Kennedy, A. L., Anthony, J. 8: Squeglia, N. (1988). Amino-acid and cDNA nucleotide sequences of human Clara cell 10 kDa protein. Biochim. Biophys. Acta, 950, 329-337.
Singh, G., Katyal, S. L., Brown, W. E., Kennedy, A. L., Singh, U. & Wong-Chong, M. L. (1990). Clara cell 10 kDa protein (CCIO): comparison of structure and function to uteroglobin. Biochim. Biophys. Acta, 1039, 348-355. Swaminathan, S., Furey, W., Pletcher, J.; Katyal, S., Singh, G. & Sax, M. (1990). Crystallization and preliminary X-ray study of rat Clara cell 10,000 && protein. J. Mol. Biol. 211, 17. Tancredi, T., Temussi, P. A. & Beato, M. (1982). Interaction of oxidized and reduced uteroglobin with progesterone. Eur. J. Biochem. 122, 101-104. Urzua, M. A., Stambaugh, R., Flinckinger, G. & Mastroianni, L. (1970). Uterine and oviduct fluid nrotein patterns in the rabbit before and after ovulaiion. Fertil. Steril. 21, 860-865. Warembourg, M., Tranchant, O., Atger, M. & Milgrom, E. (1986). Uteroglobin messenger ribonucleic acid: localization in rabbit uterus and lung by in situ hybridization. Endocrinol. (Philadelphia), 119, 1632- 1640. Weber, P. C. & Salemme, F. R. (1980). Structural and functional diversity in 4-a-helical proteins. Nature (London), 287, 82-84. Weissman, L. (1982). St,rategies for extracting isomorphous and anomalous signals. In Computational Crystallography (Sayre, D., ed.). pp. 56-63: Clarendon Press, Oxford.
by W. Hendrickson
Refined Structure of Rat Clara Cell 17 kDa Protein at 3-OA Resolution Timothy C. Urnland’?, S. Swaminathan’S, William Furey’, Gurmukh Singh’ James Pletcher’ and Martin Sax’ ‘Biocrystallography Laboratory, VA Medical Center PO Box 12055, University Dr C, Pittsburgh, PA 15240, U.S.A. and University
of
2VA Medical Center and Department of Pathology Pittsburgh School of Medicine, Pittsburgh, PA 15240, U.S.A.
(Received 3 June 1991; accepted 26 November 1991) The rat Clara cell 17 kDa protein (previously referred to as the rat Clara cell 10 kDa protein) has been reported to inhibit phospholipase A, and papain, and to also bind progesterone. It has been isolated from rat lung lavage fluid and crystallized in the space group P6,22. The structure has been determined to 3-OA resolution using the molecular replacement method. Uteroglobin, whose amino acid sequence is 55.7% identical, was used as the search model. The structure was then refined using restrained least-squares and simulated annealing methods. The R-factor is 22.5%. The protein is a covalently bound dimer. Two disulfide bonds join the monomers together in an antiparallel manner such that the dimer encloses a large internal hydrophobic cavity. The hydrophobic cavity is large enough to serve as the progesterone binding site, but access to the cavity is limited. Each monomer is composed of four a-helices. The main-chain structure of the Clara cell protein closely resembles that of uteroglobin, but the nature of many of the exposed side-chains differ. This is true, particularly in a hypervariable region between residues 23 and 36, and in the HlH4 pocket. Keywords:
Clara cell 17 kDa protein; Clara cell 10 kDa protein; uteroglobin;
amniotic fluid from rat and human, being secreted from the fetal lung (Singh et al., 1986). The rat Clara cell 17 kDa protein (RLL§) has three isotypes in the p1 range 4.6 to 50. It has been suggested that the isotypes arise from co- or post-translational modifications of a single peptide (Singh et al., 1990). RLL isotypes are covalently bonded dimers made up of two monomers of 77 residues each (Katyal et al., 1990). The monomers are linked together by two disulfide bonds in an antiparallel manner. The molecular weight of the dimer, determined from its amino acid sequence, is 16,908 daltons. Prior to the availability of sequence information, the molecular weight of the dimer was estimated to be - 10 kDa from sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Singh et al., 19853). For this reason, the protein has been referred to in the literature as
1. Introduction Clara cells are a non-ciliated, non-mucous type of secretory cell (Kuhn et al., 1974) which predominate in the distal airways. These cells have been reported to be involved in the renewal of damaged distal airway epithelium and in the metabolism of xenobiotics (Evens et al., 1978; Boyd, 1977). The Clara cell 17 kDa protein was first detected during a search for cell-specific markers for use in the study of airway cells in normal and pathological states (Singh & Katyal, 1984). The 17 kDa protein has been identified thus far in the lung lavage fluid from, and is specific to, secretory granules of Clara cells of all species examined: rat, mouse, hamster, dog, monkey and human (Singh et al., 1985a; Bedetti et al., 1987). It has also been observed in the t In partial fulfillment of the Doctor Degree. $ Author to whom all correspondence addressed.
of Philosophy should
$ Abbreviations used: RLL, Clara cell 17 kDa from rat lung lavage fluid; PLA,, phospholipase uteroglobin.
be
441 0022-2836/92/060441-08
$03.00/O
progesterone
0
1992 Academic
protein A,; UG,
Press Limited
442
T. C. Urnland
et al.
Table 1 Amino
acid sequences of RLL 5
UG RLL
and rabbit
UG
10
15
~GICPRFAHVIENLLLGTPS SSDICPGFLQVLEALLLGSES
UC RLL
20 25 SYETSLKEFEPDDTMKDAG NYEAALKPFNPASDLQNAG
UG RLL
40 M Q M K TQLKRLVDTLPQ
UG
K
RLL
KLTEKILTSPLCEQDLRV
30
K
V
45 L
K
I
V
60
L
A common numbering One-letker amino acid Glycine; H, histidine; I, glutamine; R; arginine;
T
D
35
S
L
P
50 Q
S
P
L
C
65
E
K
T T R ETRINIV
55 N
I
~~ --
~
E
70
M
M
75
-
~-
scheme is used, based on the sequence homology. code: A, alanine; C, cysteine; D, aspartate; E, glutamate; F, phenylalanine; G, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P; proline; Q, S, serine; T, threonine; V; valine; W, tryptophan; Y, tyrosine.
the Clara cell 10 kDa protein. It has been observed that RLL is an inhibitor of pancreatic phospholipase A, (PLAJ and papain, and binds progesterone (Singh et al., 1990). However, it is unknown what, if any, physiological relevance this may have. Comparison of the amino acid sequences of RLL and of rabbit uteroglobin (UG) revealed that 39 of the 70 residues (557%) contained in the UG monomer were identical to the corresponding residues in the RLL monomer (Katyal et al., 1990). The amino acid sequences of RLL and UG are given in Table 1. UG is a protein contained in the uterine washings of pregnant rabbit (Krishnan et al., 1967; Beier, 1968) and two related species, hare and pica (Nieto & Lombardero, 1982). UG has also been observed in airway cells and in the male genital tract, as well as an UG-like protein in the circulation of rabbit (Warembourg et al., 1986; Beier et al., 1975; Kikukawa & Mukherjee, 1989). It is reported to have immunosuppressive, anti-inflammatory, progesterone binding, and PLA, and protease inhibiting properties (Manjunath et al., 1984; Miele et al., 1988; Urzua et al., 1970; Levin et al., 1986; Gupta et al., 1987). It has been suggested that UG plays a role in control of inflammation and platelet aggregation, and in the regulation of cellular signal transduction (Mukherjee et al., 1988; Kikukawa & Mukherjee, 1989). Like RLL, UG is also a dimer but with 70 amino acid residues per monomer (Ponstingl et al., 1978; Popp et al., 1978), and also with two disulfide bonds between monomers. Six different crystal forms of UG have been grown (Mornon et al., 1978, 1979, 1980; Buehner & Beato, 1978), with the C222, crystal structure being solved to 1.34 A resolution (1 b = @l nm; Morize et al., 1987). Despite the amino acid sequence similarity, UG and RLL have been found to be antigenically distinct (Singh et al., 1990) as well as possess different tissue distributions. A hypervariable
region in the sequences between UG and RLL has been noted at residues 23 to 36 (Singh et al.; 1988). (The common numbering system for the sequences of RLL and UG proposed by Singh et al. (1990) is being used.) The possibly important function of RLL in the lung, together with further elucidation of its differences and similarities with UG, made a structural study . of interest. The homogeneity suggested the use of the molecular replacement method (rotation and translation functions), using UG as the model, to determine the crystal structure of RLL.
2. Materials
and Methods
(a) Crystallization RLL crystals exhibit polymorphism, dependent on f&e at which they were grown. Thus far, cubic (Swaminathan et al., 1990), monoclinic and hexagonal forms have been observed. The hexagonal crystals were grown using the sitting drop vapor diffusion method. A solution composed of 40 ~1 of 100 ,ug protein/ml (isotype III) and 5 ~1 of 60 y0 (w/v) saturated ammonium sulfate in 10 mlvr-Tris . HCl buffer (pH 7.1) was equilibrated against a reservoir solution of 60% saturated ammonium sulfate in 10 mm-Tris HCl buffer (pH 7.1). Small needle-shaped crystals grew to their maximum size (-05 mm x @05 mm x 005 mm) within a week. These crystals have space group P6522, with cell dimensions a = b = 5207 A and c = 109.27 a. Assuming a monomer weight of $454 daltons and one monomer per asymmetric unit, the Matthews (1968) coefficient was calculated to be 2.53 A”! dalton, which is in the accepted range. In this case, more than 1 monomer/asymmetric unit leads to unacceptably low Matthews coefficients (less than 127 A’/dalton): and thus the calculation is definitive. pB
(b) Data collection
and processing
X-ray diffraction data for the native crystals were collected using a Siemens multi-wire area detector. A
Structure
of Rat Clara
Table 2 Observed rejections
as a function
of resolution
%
d (4
Total
Observed
(in range)
% (cumulative)
co-46 46-39 3.9-34 34-3.0
613 358 453 602
603 346 393 381
98.4 967 868 63.3
984 97.7 94.2 850
A reflection was considered observed if I > 20(l).
Rigaku RU200 rotating anode operated at 42 kV and 65 mA with a focal spot of @2 mm x 20 mm was used to produce Ni-filtered CuK, radiation. The X-ray beam was focused using Franks mirrors. Data collection used the software described by Blum et al. (1987). Data were processed using the XENGEN package (Howard et al., 1987) and locally modified scaling programs of Weissman (1982). Although the crystals suffered substantial decay and diffracted X-rays only weakly, due to their small size, data collected from a single crystal to 3.7 A resolution were obtainable and used in the molecular replacement solution. These data were later merged with data from 4 other crystals to obtain a 3.0 A data set. The merged data set was reduced to 1733 unique reflections with I2 20(I). At 3.0 A resolution the data are 85% complete (Table Z), with a merging R-factor: of 11.7% for the 5 crystals. (c) Molecular replacement The structure of the hexagonal crystal form of RLL was solved using the molecular replacement method. Only the 3.7 A data set collected from a single crystal was used in these calculations. First, the self-rotation function (Rossmann & Blow, 1962) was calculated using the Crowther fast rotation function as implemented in ROTRAN (Crowther, 1972; Craven, 1975). Data between 50 A and 40.0 A resolution and a Patterson radius of 12.0 A were used. This only revealed the presence of 622 symmetry, with no significant peaks due to additional non-crystallographic symmetry being observed. Thus, the RLL dimer’s 2-fold axis must coincide with a crystallographic 2-fold axis, consistent with the presence of 1 monomer/asymmetric unit. The orientation of the RLL monomer in the hexagonal cell was then determined using MERLOT (Fitzgerald, 1988). Specifically, Crowther’s fast rotation function was used for a coarse search and Lattman’s rotation function (Lattman 8: Love, 1972) was used for a fine search of Eulerian space. The search model was the rabbit UG monomer from its 6222, crystal form at 1.34 A resolution (Morize et al., 1987). In this structure the UG dimer also made use of a crystallographic 2-fold axis. Cross-rotation functions were calculated using 2 different models: (1) all the atoms of the UG monomer were included; and (2) only main-chain and Cfi atoms of the UG monomer were included (the poly-alanine model). The rotation functions were calculated using 1> 3a(l) data. For the Crowther rotation function, resolution ranges of 8.0 A or 7.0 A to 3.7 A and a Patterson radius of 20 A were used. For the Lattman rotation function, resolution ranges of 8.0 A or 7.0 A to 3.9 A were ‘used. The highest peak in all of the rotation functions calculated, using both models, consistently was at or near the Eulerian angles a = 30.0”,
Cell 17 LDa Protein
443
fi = 81.0” and y = 1800” (as defined by MERLOT). The UG search models’ original orientations were such that their dimer 2-fold axes were parallel to the RLL hexagonal cell’s b-axis at the Euler angles a = /l = y = 0”. Thus, this solution of the rotation function indicates that the UG search models had been orientated in the RLL cell such that their dimer 2-fold axes lay parallel to the crystallographic 2-fold axis in the [- 1 1 0] direction. This result is consistent with that of the self-rotation function. The translation problem of the molecular replacement method was solved using an R-factor minimum search, where:
R = W’ol -JW’,II/W’~I> calculated by RMAP (W. Furey, unpublished results). The R-factor search was also used at the same time to determine the correct space group. The Laue symmetry was known to be 6/mmm, but due to the limited number of observed 001 reflections and the enantiomorphic nature of the space group possibilities, the space group could not be determined unambiguously by the systematic absences. Thus, the R-factor search had to be conducted in each of the 6 possible space groups. The fact that the dimer’s 2-fold axis coincides with a crystallographic 2-fold axis restricted the dimer’s center of mass to lie on a given line residing on specific planes for each possible space group. From the orientation of the search model in the RLL cell given by the rotation function, the R-factor minimum search was reduced to a search over the set of fractional co-ordinates of {z, --2, z}, where z = 90, 5/12, 516, l/4, l/6 and l/12 for the space group P622, P6,22, P6,22, P6,22, P6,22 and P6,22, respectively. Prior to the R-factor minimum search, the UG dimer co-ordinates were translated so that its center of mass was at the origin, rotated to the optimum orientation as determined from the rotation function, and then translated along z to the value appropriate for each search. The search model was then reduced to only one monomer containing only main-chain and CB atoms, which was translated along the line (2, --2, z}. The minimum value of R ( =5@8%) was obtained in space group P6,22, with the poly-alanine UG monomer positioned such that the center of mass of the dimer was at x = 0.575, y = -0575 and z = l/12. The minimum R-factors obtained for the other 5 possible space groups ranged between 57.5 ye and 68.8%. The results of the R-factor searches are given in Table 3. All observed reflections from the 3.7 a data set were used. This initial orientation and position of the UG polyalanine monomer model in the P6,22 RLL cell was then
Table 3 Minimum R-factors calculated for the orientated UG poly-alanine monomer search model in each of the possible space groups Space group P622 P6,22 P6,22 P6,22 P6,22 P6,22
Min. Et at: 06882 05865 0.5750 06544 05940 0.5077
z
Y
z
0.500 0575 0575 0450 0%50 0.575
- 0500 - 0.575 -0.575 -0.475 - 0.856 - 0.575
PO, l/2 5/12, 11/12 5/6, l/3 l/4, 3/4 l/6, 2/3 l/12, 7/12
The fractional co-ordinates give the position of the dimer’s center of mass at which the minimum R-factpr is obtained. The 2 different values of z for each space group correspond to shifts of the origin by l/2 unit cell along the c-axis.
t R = UFol -W’cll/Wol.
T. 6. Umtand
444
refined by least-squares using the rigid-body refinement program GREF (Furey, 1990). For the first 11 cycles, the model was treated as a single rigid body. Then for cycles I2 through 17 the model was broken up into 4 rigid bodies, with each roughly corresponding to one of the 4 cc-helices contained in the UG monomer. The 4 rigid bodies were UG residues 1 to 15; 16 to 29, 30 to 48 and 49 to 70. This gave R = 45.1 To for 1046 reflections to 3.7 d resolution, with I > 3@). (d) Model building
and rejhement
The refined co-ordinates of the poly-alanine UG search model were used to calculate the initial phases of a 2F0 - F, map, with the 1085 observed reflections to 3.7 d having FO> 2a(FO). Density corresponding to that expected for the large side-chains and disulfide bonds of RLL was observed. This confirmed the correctness of the solution, as only CB side-chain atoms had been included in the phase calculations. The RLL sequence was then built into the 2F,-F, map using the FRODO/TOM software (Jones, 1982; Cambillau & Horjales, 1987) on a Silicon Graphics Personal Iris computer. This first RLL model led to an R = 41.4%. Refinement was then begun using GPRLSA (Furey et al., 1982), which is a modified version of Hendrickson’s restrained least-squares refinement package PROLSQ (Hendrickson & Konnert, 1980). Initially, only the 3.7 A data set was used, but later data from all 5 crystals were merged into a single data set and included in the refinement. Cycles of refinement were alternated with manual corrections of the model using 2FO-- Fc and F,- F, maps, and occasionally residuedeleted 2F,- F, maps. In the later stages of refinement, a modified version of GPRLSA was used that restrained the 2 disulfide bonds that exist between monomers. This modification was necessary since GPRLSA will not normally restrain bonds that cross an asymmetric unit boundary. When the R-factor had reached 3@8% for 3.0 a data, refinement was continued using XPLOR (Bruenger et al., 1987). After the use of XPLOR, the modified version of GPRLSA was used again to restore the disulfide bonds to reasonable geometry, since XPLOR is also incapable of dealing correctly with disulfide bonds spanning asymmetric units. The final R-factor was 22.5o/o for 1661 observed reflections in the resolution range
et al. !@O A to 3.0 8, and the deviations from ideal geometry were acceptable (Table 4). Due to the lack of high resolution data, only an overall thermal factor was used, where B3= 7.23 8’. Eo solvent molecules, except for a singie sulfate ion, have been placed in the model. The electron density, which has been modeled by a sulfate ion, lies between the side-chain amino groups of Lys62 and the 2-fold symmet’ry-related residue in an adjoining dimer. In the final 2F,-F, map, the only residues for which the main-chain was not well-defined were Srg74 and Val75. ,4s a check that the final RLL model was not overly biased by the initial UG model, residue-deleted 2F0--F, maps were calculated. Specifically, a 5 residue segment in the RLL model was deleted from the phase calculation if t,he side-chain of the central residue in the corresponding segment in UG had a greatly different size or shape. The maps were then viewed using FRODO/TOM, and density for all of the deleted residues was as expected for the RI& sequence. Also, the RLL chain extends further on both the h’ and C-terminal ends than does UG, and residuedeleted maps confirmed the positions of these residues. The RLL refined monomer co-ordinates and structure factors have been deposited at the Brookhaven Protein Data Bank, with the reference codes 1CCD and RICCDSF.
3. Results and Discussion The native P6,22 form of RLL is a covaientl?; bound dimer that has exact (crystallographic) 2-fold symmetry between its monomers (Fig. 1). Two disulfide bonds, Cys3 to Cys69’ and Cys69 to CysJ’, join the monomers together in an antiparallel manner. (Residues contained in the second monomer of the dimer are indicated with a prime.) Only two hydrogen bonds act to stabilize the dimer. These are between Gln40 and Thr52’ and the symmetry-relat,ed pair. It would appear that in addition to the disulfide bonds, hydrophobic forces are important in maintaining the dimer. The
Table 4 Deviations from ideal geometry restrained refinement
after
0
r.m.s. deviation
A. Distance restraints (A)
Bond distance Angle Planar Planar Chiral
distance l-4 distance restraint volume (A3)
B. Non-bonded contacts (A) Single torsion Multiple torsion Possible H-bond C. Torsion angles (deg.) Planar Staggered ( + / - 60, 180”) Orthonormal ( + / - 90”) r.m.s., root-mean-square.
0.020 0.045 0.035 0.020 0.200
0.016 0.062 0040 0.009 0.284
0300 0300 0300
0216 0.248 0174
50 150 150
2.6 26.3 322
Figure 1. A ribbon cartoon (Priestle, 1988) of the RLL dlmer viewed down the crystallographic 2-fold axis. The 2 disulfide bonds between the monomers are indicated by bold continuous lines.
Structure of Rat Clara Cell 17 kDa Protein
Figure 2. The C” skeleton of the RLL monomer. The residue labels follow the common numbering scheme for RLL and UG (Singh et al., 1990).
monomer is composed of four a-helices, as shown in Figure 2. However, the monomer does not exhibit the four a-helical bundle motif (Weber & Salemme, 1980) as the helices of the two monomers composing the dimer interact to create a large internal cavity. Helix 1 encompasses Pro4 through Leu14. A /?-turn (Leul3 to Serl7) leads into helix 2 (Glu18 to Lys26). Following the second helix is another b-turn consisting of Lys26 to Asn29. Helix 3 extends between Ser32 and Thr47, and helix 4 ranges from Gln50 to Leu64. No /?-structure is present. The chain ends have no particular secondary structure. The five proline residues present in the monomer tend to break the structure into different segments. The main-chain dihedral angles for the helical regions had mean values of (4) = - 61.0” and (9) = -41.4”. All main-chain dihedral angles are in allowed regions of conformational space. Asn29 lies in the left-handed helical region of the Ramachandran map (Ramachandran et al., 1963).
445
The corresponding residue in UG, Glu29, also has this conformation. The root-mean-square deviation between main-chain atoms of residues 1 to 70 of RLL and UG is @SO8. A large hydrophobic cavity exists between the two monomers of the dimer. The residues that define this cavity are Phe6, Va19, LeulO, Leul3, Leu14, Tyr21, Leu25, Gly38, Leu41, Va145, Ile56, Leu59, Thr60, Ile63 and the symmetry-related residues on the second monomer. The distances between residues lining the cavity and the corresponding residues related by 2-fold symmetry give an indication of the width of the cavity. For example, the distance between Thr60 Cyl and Thr60’ CY1 is 1@18, and between Leu25 Cy and Leu25’ Cy is 23.6 8. The length of the cavity along the 2-fold axis may be approximated by the distance between Phe6 CE2and Va145’ Cy2’, which is 13.5 8. However, it appears that the cavity is not highly accessible to the outside environment (Fig. 3). This cavity is very similar to that of UG, both in geometry and in residue make-up. Of the 14 residues per monomer that line the cavity, 11 are identical between UG and RLL. Conservative substitutions have occurred for the three residues which differ. It has been suggested that the large internal cavity contained within UG is the binding site for progesterone based on its hydrophobic nature and geometry (Mornon et al., 1980; Morize et al., 1987). By analogy, the cavity within RLL may be suspected of being the progesterone binding site as well. However, these sites are controversial due to limited access to the cavities. In addition, only the reduced form of either protein binds progesterone, while the native proteins are in the oxidized form that exhibits little or no binding. No oxidized UG-hormone complex could be isolated from the uterus (Tancredi et al., 1982). Nevertheless, model building demonstrated that the dimensions of the interior cavity of RLL would allow a progesterone molecule to reside there, analogous to the proposed UG-progesterone model, if several side-chains moved slightly from their positions in the native
Figure 3. A stereo view of the RLL dimer with all atoms included. The view is normal to the 2-fold axis. Notice that there is limited access to the interior cavity.
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Figure 4. Stereo view of the RLL interior cavity with a molecule of progesterone modeled inside. The view is down the dimer’s Z-fold axis. Only the amino acid residues that line the cavit,y are displayed (continuous lines), except Phe6 and PheS’, which have been omitted for clarity. The progesterone (broken lines) has been fitted according to the proposed UG-progesterone model.
structure (Pig. 4). The closest contact is bet,ween Cd1 of Leul3 and Cl9 of the hormone, at 1.8 A. Side-chains of LeulS’, Leu41 and Leu41’ contact the hormone at distances of 2.0 to 2.5 A, and those of LeulO, LeulO’ and Ile56 are 2.5 to 3.0 A away. The only close contact involving a main-chain atom of les$ than 4.0 A is between the carbonyl 0 of Ile56 and C21 of progesterone, at 3.2 A. Hydrogen bonds may exist between Oq of Tyr21 and TyRl’ of RLL and 03 and 020 of progesterone, as in the UG-progesterone model. Atomic co-ordinates for progesterone were taken from Campsteyn et al. (1972). No electron density was observed which could be attributed to bound progesterone in the hydrophobic cavity or at any other site in the RLL crystal structure. However, since no progesterone was added to the mother liquor, this absence of electron density only demonstrates that the RLL was isolated and crystallized in the unbound state; and does not disprove the internal cavity binding site hypothesis. If the cavity is the binding site, then lack of access to the cavity in the oxidized RLL structure would correlate with its non-binding of the hormone. The reduction of the protein’s disulfide bonds may allow the two monomers to partially separate, and thus form an access channel into the cavity for the hormone. Clearly the structure of the protein-hormone complex needs to be determined to provide added insight regarding the binding site.
ends of the dimer form a’ The C and N-terminal pocket. This is separated from the large internal cavity by Phe6 and Phe6’, but’ a rotation of the phenyl groups would open a small channel into the internal cavity. This pocket is deeper in RLL than the corresponding pocket in UG, due to the additional residues contained at both ends of RLL. RLL possesses surface pockets that are formed by helix 1 and helix 2 (HlH2) and by helix 1 and helix 4 (HlH4), as does UG. HlH2 is formed by Gly5, Gln8, Va,l9, Ala24, Lys26, Pro27 and Phe28. HlH4 is formed by Aspl, Ile2, Leu7? Gln8, LeulO, Glull, Va157, Thr60, Glu61, Leu64 and Thr65. Although the main-chain conformation of these pockets is similar for both UG and RLL, the residue compositions are different. For HlH2, only two of the seven residues that form the pocket are ident,ical in both proteins. Likewise, for HlH4, only four of the 11 residues in the pocket are identical. The HlH2 pocket of RLL has one basic and no acidic residues, whereas UG has three basic and one acidic residue. The HlH4 pocket of RLL has three acidic and no basic residues, and UG has two basic and two acidic residues in the same pocket. Thus? the pockets of the two proteins differ in both charge and hydropathy. Also, the HlH4 pocket of RLL is smaller than that of UG. This is due to the substitution iu RLL of a leucine for an alanine at residue seven, and a different main-chain geometry near the N
Figure 5. A stereo view of the superimposed amino acid residues from RLL and UG that form the HlH4 pocket. For RLL (continuous lines) these residues are Aspl, Ile2, Leu7, Glu8, LenlO, Glul I, Va157, Thr60, GluGI, Leu64 and Thr65. For UG (broken lines) these residues are Glyl, Ile2, Ala7, Hi&, IlelO, Glul I. Met57, Thr60, Glu61, Va164 and Lys65. The RLL pocket is smaller mainly due to the substitution of leucine for alanine at residue 7.
Structure
of Rat Clara
terminus causing the side-chain of Ile2 of RLL to project further out into the pocket than the corresponding side-chain in UG (Fig. 5). Preliminary nuclear magnetic resonance studies have indicated that interaction between progesterone and oxidized UG in solution caused conformational changes localized to the HlH4 pocket region (Jamin et al., 1989). If this conformation change is relevant to hormone binding, then perhaps the decreased ability of RLL to bind progesterone as compared to UG (Singh et al., 1990) is due to the differing properties of the HlH4 pockets in the two proteins. Singh et al. (1990) reported the presence of a hypervariable region in the RLL and UG amino acid sequences between residues 23 and 36, where only 4 of the 14 residues are identical. However, the main-chain conformation in this region is similar for both proteins, containing the carboxyl end of helix 2, the amino end of helix 3, and the turn connecting the helices. Again, RLL and UG differ in the number of charged side-chains each contains in this region. RLL has one basic and one acidic residue, and UG has two basic and five acidic residues in this section. Much of this region is exposed to solvent and so it may give rise to the functional differences
between the two proteins. We thank Dr J. P. Mornon for kindly providing us with the atomic co-ordinates of UG. This work was supported by the U.S. Army Medical Research and Development Command under Intragovernmental order no. 87PP7852. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army. An Andrew Mellon Predoctoral Fellowship sponsored by the A. W. Mellon Educational and Charitable Trust and administered by the University of Pittsburgh was held by T.C.U. References Bedetti, C. D.; Singh, J., Singh, G.; Katyal, S. L. & Wong-Chong, M. L. (1987). Ultrastructural localization of rat Clara cell 10 KD secretory protein by the immunogold technique using polyclonal and monoelonal antibodies. J. Histochem. Cytochem. 35, 789-794. Beier, H. M. (1968). Uteroglobin: a hormone-sensitive endometrial protein involved in blastocyst development. Biochim. Biophys. Acta, 160, 2899291. Beier, H. M., Bohn, H. & Muller, W. (1975). Uteroglobin-like antigen in the male genital tract secretions. Cell Tiss. Res. 165, l-11. Blum, M., Metcalf; P., Harrison, S. C. 85 Wiley, D. C. (1987). A system for collection and on-line integra’tion of X-ray diffraction data from a multiwire area detector. J. Appl. Crystallogr. 20, 235-242. Boyd, M. R. (1977). Evidence for the Clara cell as a site of cytochrome P450-dependent mixed-function oxidase activity in lung. Nature (London), 269, 713-715. Bruenger, A. T., Kuriyan, J. & Karplus, M. (1987). Crystallographic R factor refinement by molecular dynamics. Science, 235, 4588460. Buehner, M. & Beato, M. (1978). Crystallization and preliminary crystallographic data of rabbit uteroglobin. J. Mol. Biol. 120, 337-341. Cambillau, C. & Horjales, E. (1987). TOM: a FRODO subpackage. J. Mol. Graph. 5, 174.
Cell 17 IcDa Protein
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Campsteyn, H., DuPont, L. & Dideberg, 0. (1972). Structure cristalline et moleculaire de la progesterone CzlH,,O,. Acta Crystallogr. sect. B, 28, 3032-3042. Craven, B. M. (1975). Computer programs ROTRAN for the determination of crystal structures which contain rigid body fragments of known structure. Univ. of Pittsburgh, Dept. of Crystallography, Technical Report TR-75-2. Crowther, R. A. (1972). The fast rotation function. In The Molecular Replacement Method (Rossmann, M. G., ed.), pp. 173-178, Gordon and Breach, New York. Evens, M. J., Cabral-Anderson, L. J. & Freeman, G. (1978). Role of Clara cell in renewal of the bronchiolar epithelium. Lab. Invest. 38, 648-655. Fitzgerald, P. M. D. (1988). MERLOT, an integrated package of computer programs for the determination of crystal structures by molecular replacement. J. Appl. Crystallogr. 21, 273-278. Furey, W. (1990). PHASES-a program package for the processing and analysis of diffraction data from macromolecules. American Crystallographic Association Fortieth Anniversary Meeting, New Orleans, LA, abstract PA33. Furey, W., Wang, B. C. & Sax, M. (1982). Crystallographic computing on an array processor. J. Appl. Crystallogr. 15, 160-166. Gupta, R. P., Patton, S. E.: Jetten, A. M. & Hook, G. E. R. (1987). Purification, characterization and proteinase-inhibitory activity of a Clara cell secretory protein from the pulmonary extracellular lining of rabbits. Biochem. J. 248, 337-344. Hendrickson, W. A. & Konnert, J. H. (1980). Stereochemically restrained crystallographic leastsquares refinement of macromolecule structures. In Biomolecular Structure, Conformation, Function and Evolution (Srinivasan, R., ed.), vol. 1, pp. 43-57, Pergamon Press, Oxford. Howard, A. J., Gilliland, G. L., Finzel, B. C. & Poulos, T. L. (1987). The use of an imaging proportional counter in macromolecular crystallography. J. Appl. Crystallogr. 20, 383-387. Jamin, N., Roy, P., Fridlansky, F., Delepierre, M., Milgrom, E., Roques, B. P. & Mornon. J. P. (1989). Preliminary assignments of the aromatic and some methyl group resonances of the ‘H-NMR spectrum of bhe oxidized form of uteroglobin. Eur. J. Biochem. 183, 219-226. Jones, T. A. (1982). FRODO: a graphics fitting program for macromolecules. Computational In Crystallography (Sayre, D., ed.), pp. 3033317, Clarendon Press, Oxford. Katyal, S. L., Singh, G., Brown, W. E., Kennedy, A. L., Squeglia, N. & Wong-Chong, M. L. (1990). Clara cell secretory (10 k daltons) protein: amino acid and oDNA sequences, and developmental expression. Prog. Respir. Res. 25, 29-35. Kikukawa, T. & Mukherjee, A. B. (1989). Detection of a uteroglobin-like phospholipase A, inhibitory protein in the circulation of rabbits. Mol. Cell. Endocrinol. 62, 177-187. Krishnan, R. S. & Daniel, J. C. Jr (1967). “Blastokinin”: inducer and regulator of blastocyst development in the rabbit uterus. Science, 158, 490-492. Kuhn, C. III, Callaway, L. A. & Askin, F. B. (1974). The formation of granules in the bronchiolar Clara cells of the rat. J. Ultrastruct. Res. 49, 387-400. Lattman, E. E. $ Love, W. E. (1972). A rotational search procedure for detecting a known molecule in a crystal. Acta Crystallogr. sect. B, 26, 1854-1857.
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Levin, S. W., Butler, J. DeB., Schumacher, U. K., Wightman, P. D. & Mukherjee; A. B. (1986). Uteroglobin inhibits phospholipase A, activity. Life sci. 38, 1813-1819. Manjunath, R., Chung, S. I. & Mukherjee, A. B. (1984). Crosslinking of uteroglobin by transglutaminase. Biochem. Biophys. Res. Commun. 121, 400-407. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Miele, L., Cordella-Miele, E., Facchiano, A. & Mukherjee, A. B. (1988). Novel anti-inflammatory peptides from the region of highest similarity between uteroglobin 335, 726-730. and lipocortin I. Nature (London), Morize, I., Surcouf, E.; Vaney, M. C., Epelboin, Y., Buehner, M., Fridlansky, F., Milgrom, E. & Mornon, J. P. (1987). Refinement of the C222, crystal form of oxidized uteroglobin at 1.34A resolution. J. Mol. Biol. 194, 725-739. Mornon, J. P., Surcouf, E., Bally, R., Fridlansky, F. & Milgrom, E. (1978). X-ray analysis of a progesteronebinding protein (uteroglobin): preliminary results. J. Mol.
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Mornon, J. P., Bally, R., Fridlansky, F. & Milgrom, E. (1979). Characterization of two new crystal forms of uteroglobin. J. Mol. Biol. 127, 237-239. Mornon, J. P., Fridlansky, F., Bally, R. & Milgrom, E. (1980). X-ray crystallographic analysis of a progesterone binding protein. The 6222, crystal form of oxidized uteroglobin at 2.2 A resolution. J. Mol. Biol. 137, 415-429. Mukherjee, A. B.; Cordella-Miele, E., Kikukawa, T. & Miele, L. (1988). Modulation of cellular response to antigens by uteroglobin and transglutaminase. In Advances in Exp. Med. and Bio.: Advances in Post-translational Modifications of Proteins and Aging
(Zappia, V., Galletti, P., Porta, R. 85 Wold, F., eds), vol. 231, pp. 135152, Plenum Press, New York. Nieto, A. & Lombardero, M. (1982). Uteroglobin-like antigens in species of Lagomorpha. Comp. Biochem. PhysioE. B, 71, 511-514. Ponstingl, H., Nieto, A. & Beato, M. (1978). Amino acid sequence of progesterone-induced rabbit uteroglobin. Biochemistry, 17, 390883912. Popp, R. A., Foresman, K. R., Wise, L. D. & Daniel, J. C., Jr (1978). Amino acid sequence of a progesterone-binding protein. Proc. Nat. Acad. Sci., U.S.A. 75, 5516-5519. Priestle, J. P. (1988). RIBBON: a stereo cartoon drawing program for proteins. J. Appl. Crystallogr. 21, 572-576. Ramachandran, G. N., Ramakrishnan, C. & Sasisekharan, V. (1963). Stereochemistry of polypeptide chain configurations. J. Mol. Biol. 7, 95-99.
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Rossmann, M. G. & Blow, D. M. (1962). The detection of subunits within the crystallographic asymmetric unit. Acta Crystallogr. 15, 24-31. Singh, G. & Katyal, S. L. (1984). An immunologic study of the secretory products of rat Clara cell. J. Histochem.
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Singh, G., Katyal, S. L., Brown, W. E., Kennedy, A. L., Singh, U. & Wong-Chong, M. L. (1990). Clara cell 10 kDa protein (CCIO): comparison of structure and function to uteroglobin. Biochim. Biophys. Acta, 1039, 348-355. Swaminathan, S., Furey, W., Pletcher, J.; Katyal, S., Singh, G. & Sax, M. (1990). Crystallization and preliminary X-ray study of rat Clara cell 10,000 && protein. J. Mol. Biol. 211, 17. Tancredi, T., Temussi, P. A. & Beato, M. (1982). Interaction of oxidized and reduced uteroglobin with progesterone. Eur. J. Biochem. 122, 101-104. Urzua, M. A., Stambaugh, R., Flinckinger, G. & Mastroianni, L. (1970). Uterine and oviduct fluid nrotein patterns in the rabbit before and after ovulaiion. Fertil. Steril. 21, 860-865. Warembourg, M., Tranchant, O., Atger, M. & Milgrom, E. (1986). Uteroglobin messenger ribonucleic acid: localization in rabbit uterus and lung by in situ hybridization. Endocrinol. (Philadelphia), 119, 1632- 1640. Weber, P. C. & Salemme, F. R. (1980). Structural and functional diversity in 4-a-helical proteins. Nature (London), 287, 82-84. Weissman, L. (1982). St,rategies for extracting isomorphous and anomalous signals. In Computational Crystallography (Sayre, D., ed.). pp. 56-63: Clarendon Press, Oxford.
by W. Hendrickson