.J. 3101. &I.
(1992) 228, X-251
Crystal Packing in Six Crystal Forms of Pancreatic Ribonuclease Marie-Pierre Crosio, Jo51 Janin and Magali Jullien Laboratoire de Biologic Structuralr UMR 9920 CXRS Mt.
433 Cniz?ersith Pa,ris-l\‘ud 91405~Orsay, France
(Received 6 March
1992;
accepted 10 July
1992)
We compare the molecular packing of bovine pancreatic ribonuclease A (R?;ase -A) in six cry&al forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which burv 1570 A2 t)o 2790 !I’ of the RSase surface. Regions of the protein surface involved in the six packings cover almost’ the whole R’Nase molecule. The abundance of polar interactions, about) one per 200 A2. is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large int’erface covering 1800 a2 and eight to ten polar interactions. Tts presence in the three salt-grown crystal forms suggests t’hat it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms cont’ain only small interfaces, implying a different mechanism of nucleation. Krywords:
ribonuclease; crystal packing: protein association
1. Introduction The way promin molecules associate regularly to yield crystals is governed by weak packing forces, which are modulated by precipitating agents. Depending on the precipitating agent and details of the experimental procedure, the same protein may yield several crystal forms, where the molecule has while making essentiallv the same structure different ‘interactions with its neighbours. A comparison of the molecular packing in crystal structures that have been solved to high resolution and for which atomic co-ordinates are available should yield insight on the role of the precipitant and the nature of the packing forces. Commonly used precipitants fall into three categories with distinctive physicochemical properties: salts, organic solvents and polyols (McPherson, 1990). Ali three types have been used to crystallize bovine pancreatic ribonuclease A (Rxase A) or it,s derivabives. We examine here six crystal forms of this protein grown under different conditions. Two are from alcohol solutions: a monoclinic form of native RNase A and an orthorhombic form of the product of a chemical cross-link within the protein. The monoclinic form grows in 43% t-but,anol. It is one of some 14 crystal forms of the native protein obtained with organic solvents (King et al., 1956, 1962). Its structure is known to very high resolution
(Borkakoti et 01.. 1982; Wlodawer et al., 1982; Campbell & Petsko. 1987; Wlodawer et al.. 1988). The cross-linked protein. called here RNase X, was crystallized in 30 7~ ethanol and its structure was solved by Weber el al. (1985). Three other crystal forms are from high salt: a trigonal form of RNase 8, an orthorhombic form of a thymidine adduct named here RNase T-H,, and a triponal form of it semisynthetic RNase A, which we shall call Rh’asr A*. R’xasr S, obtained by lirnited proteolysis of RSase A with subtilisin, crystallizes in 400/,, ammonium sulphahe and 3 x-caesium chloride. Tts structure was solved by Wyckoff it rrl. (1970). RSasr T-H,, is a derivative of ribonuclease A with a, thymidine covalently bound to HislP. It also sulphate-caesium crystallizes in ammonium chloride. yielding orthorhombic crystals with two prot.ein molecules in the asymmetric unit,. I& structure was solved by ?;achman rt nl. (1990). RNase A* is a cat,alytically act’ive. noncovalent complex of residues 1-118 with the synthetic peptide 111-124. It crystallizes in ammonium sulphateecaesium chloride. The X-ray structure of several variant’s has been determined by Martin et nl. (1987) and Demel et al. (1992). Last, RNase N, a glycosylated form of ribonuclease .A. has been crystallized from polyethylene glycol and analysed by Williams et al. (1987). It is monoclinic and has two protein molecules in its asymmetric unit.
‘I’lw wystal packing in monoc~linic~ KNase A ha> Jwfw cwmpared to t)hat of trigonal K?iase S ((‘rosin, (,t ~1.. 1990) anti of orthorhombic TLSase T-H,, (Svcwison it al.. 1991). Observed differences coulti Iw at,tril)utwi eitht,r to t’ht cahemical modification of’ the protein or to the nature of thr precipitant. \\‘hen more wystal forms are wnsitlered. somc~ of the cwnc~lusions dra,wn in earlier studies must hi reviwtl. So systematic different is seen in alwhogrown wrsu.s salt -grown carvst als in either the tot d arra of the prot’ein surfaw I)urie(i in w-yst’al cwntacts or the total number of polar intwwtions. The various c-hrmical modificxtions ih1)pt’klr’ un(‘onst‘qurnt~ial. ‘l’hr two types difier. howt~ver. in the numlw size an(l of pairwiscl intrrfacw. Alwho-grown crystals wnt,ain a large number. of small interfaces. In salt-grown yystals, t,hr rihonuclease molec~ules form symrnet,riwI dimers. whicalr arc. likeI?- intermetiia,tes in c~rystallization with salt.
2. Methods Atomic co-ordinates (Table I) are from the Brookhaven Protein Data Bank (Bernst,ein rt al., 1977). Ligands Wtlrt‘ incaludrd in all calculations when present in the CYordinate sets. When more than one side-(
(1992) 228, X-251
Crystal Packing in Six Crystal Forms of Pancreatic Ribonuclease Marie-Pierre Crosio, Jo51 Janin and Magali Jullien Laboratoire de Biologic Structuralr UMR 9920 CXRS Mt.
433 Cniz?ersith Pa,ris-l\‘ud 91405~Orsay, France
(Received 6 March
1992;
accepted 10 July
1992)
We compare the molecular packing of bovine pancreatic ribonuclease A (R?;ase -A) in six cry&al forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which burv 1570 A2 t)o 2790 !I’ of the RSase surface. Regions of the protein surface involved in the six packings cover almost’ the whole R’Nase molecule. The abundance of polar interactions, about) one per 200 A2. is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large int’erface covering 1800 a2 and eight to ten polar interactions. Tts presence in the three salt-grown crystal forms suggests t’hat it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms cont’ain only small interfaces, implying a different mechanism of nucleation. Krywords:
ribonuclease; crystal packing: protein association
1. Introduction The way promin molecules associate regularly to yield crystals is governed by weak packing forces, which are modulated by precipitating agents. Depending on the precipitating agent and details of the experimental procedure, the same protein may yield several crystal forms, where the molecule has while making essentiallv the same structure different ‘interactions with its neighbours. A comparison of the molecular packing in crystal structures that have been solved to high resolution and for which atomic co-ordinates are available should yield insight on the role of the precipitant and the nature of the packing forces. Commonly used precipitants fall into three categories with distinctive physicochemical properties: salts, organic solvents and polyols (McPherson, 1990). Ali three types have been used to crystallize bovine pancreatic ribonuclease A (Rxase A) or it,s derivabives. We examine here six crystal forms of this protein grown under different conditions. Two are from alcohol solutions: a monoclinic form of native RNase A and an orthorhombic form of the product of a chemical cross-link within the protein. The monoclinic form grows in 43% t-but,anol. It is one of some 14 crystal forms of the native protein obtained with organic solvents (King et al., 1956, 1962). Its structure is known to very high resolution
(Borkakoti et 01.. 1982; Wlodawer et al., 1982; Campbell & Petsko. 1987; Wlodawer et al.. 1988). The cross-linked protein. called here RNase X, was crystallized in 30 7~ ethanol and its structure was solved by Weber el al. (1985). Three other crystal forms are from high salt: a trigonal form of RNase 8, an orthorhombic form of a thymidine adduct named here RNase T-H,, and a triponal form of it semisynthetic RNase A, which we shall call Rh’asr A*. R’xasr S, obtained by lirnited proteolysis of RSase A with subtilisin, crystallizes in 400/,, ammonium sulphahe and 3 x-caesium chloride. Tts structure was solved by Wyckoff it rrl. (1970). RSasr T-H,, is a derivative of ribonuclease A with a, thymidine covalently bound to HislP. It also sulphate-caesium crystallizes in ammonium chloride. yielding orthorhombic crystals with two prot.ein molecules in the asymmetric unit,. I& structure was solved by ?;achman rt nl. (1990). RNase A* is a cat,alytically act’ive. noncovalent complex of residues 1-118 with the synthetic peptide 111-124. It crystallizes in ammonium sulphateecaesium chloride. The X-ray structure of several variant’s has been determined by Martin et nl. (1987) and Demel et al. (1992). Last, RNase N, a glycosylated form of ribonuclease .A. has been crystallized from polyethylene glycol and analysed by Williams et al. (1987). It is monoclinic and has two protein molecules in its asymmetric unit.
‘I’lw wystal packing in monoc~linic~ KNase A ha> Jwfw cwmpared to t)hat of trigonal K?iase S ((‘rosin, (,t ~1.. 1990) anti of orthorhombic TLSase T-H,, (Svcwison it al.. 1991). Observed differences coulti Iw at,tril)utwi eitht,r to t’ht cahemical modification of’ the protein or to the nature of thr precipitant. \\‘hen more wystal forms are wnsitlered. somc~ of the cwnc~lusions dra,wn in earlier studies must hi reviwtl. So systematic different is seen in alwhogrown wrsu.s salt -grown carvst als in either the tot d arra of the prot’ein surfaw I)urie(i in w-yst’al cwntacts or the total number of polar intwwtions. The various c-hrmical modificxtions ih1)pt’klr’ un(‘onst‘qurnt~ial. ‘l’hr two types difier. howt~ver. in the numlw size an(l of pairwiscl intrrfacw. Alwho-grown crystals wnt,ain a large number. of small interfaces. In salt-grown yystals, t,hr rihonuclease molec~ules form symrnet,riwI dimers. whicalr arc. likeI?- intermetiia,tes in c~rystallization with salt.
2. Methods Atomic co-ordinates (Table I) are from the Brookhaven Protein Data Bank (Bernst,ein rt al., 1977). Ligands Wtlrt‘ incaludrd in all calculations when present in the CYordinate sets. When more than one side-(
