- Email: [email protected]
Mitochondrial creatine kinase - a square protein
811
Mitochondrial creatine k i n a s e - a square protein Wolfgang Kabsch* and Karin Fritz-Wolf The recently determined structure of octameric mitochondrial creatine kinase has provided new insights into the functioning of this enzyme and its role in channelling energy from the mitochondria to the cytoplasm. Creatine kinase, a member of the family of guanidino kinases, is structurally similar to glutamine synthetase, suggesting a possible evolutionary link between both protein families.
Addresses Max-Planck-lnstitut for medizinische Forschung, Abteilung Biophysik, Jahnstrasse 29, D-69028 Heidelberg, Germany *e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:811-818
Mi-CKs are found exclusively in the mitochondrial intermembrane compartment, being attached to the inner membrane, but only the octameric form can interact with both the inner and outer rnitochondrial membranes [9"]. Mi-CK is functionally and perhaps also physically coupled to the adenine nucleotide translocator [10"'] of the inner membrane and to porin [11[ of the outer membrane. This enables the enzyme to utilise the intramitochondrially produced ATP for synthesis of PCr, which is exported into the cytosol for regeneration of ATP by the cytosolic isoforms. This interplay between the cytosolic CKs and the Mi-CKs, with the easily diffusible (P)Cr serving as an energy shuttle system between the cellular compartments, is referred to as the 'PCr circuit' [3,12,13].
http://biomednet.com/elecref/0959440X00700811 © Current Biology Ltd ISSN 0959-440X Abbreviations CK creatine kinase Cr creatine Mi-CK mitochondrial CK Mib-OK mitochondrial basic CK Per phosphocreatine rmsd root mean square deviation TSAC transition state analogue complex
Introduction Excitable cells and tissues such as muscle, brain, or spermatozoa consume large amounts of energy in a fluctuating manner. During activation, the required energy, in the form of ATP, is continuously and efficiently regenerated from a large pool of phosphocreatine (PCr) by the reaction PCr2-+ MgADP-+ H+-+MgATPZ-+ Cr catalysed by the enzyme creatine kinase (CK; EC 2.7.3.2; for reviews sec [1-3]). The reaction, believed to occur by a direct inline transfer of the phosphoryl group between the bound substrates [2,4,5], is accompanied by a conformational change of the enzyme [1,6,7"]. In addition to productive complexes, CK also forms a stable dead-end inhibition complex with nitrate, MgADE and Cr which mimics the transition state of the enzyme [8]. CK exists in tissue-specific (M, muscle-type; B, braintype), as well as in compartment-specific (cytosolic and mitochondrial), isoforms. T h e cytosolic forms are coupled to glycolysis to produce PCr, or are specifically associated at sites of high-energy consumption to produce ATE or balance the PCr/Cr and ATP/ADP pools in the cytosol. In contrast to the cytosolic forms, which are always homo- or heterodimeric (MM-, BB- or MB-CK), the mitochondrial creatine kinases (Mi-CKs) also form octamers comprising four dimers as stable building blocks. T h e mitochondrial isoforms are functionally coupled to oxidative phosphorylation to produce PCr.
Mi-CKs are also expressed in a tissue-specific manner and occur in two isoforms: a striated muscle-specific, 'sarcomeric' Mia-CK (b standing for basic); and the so-called 'ubiquitous', more acidic (a) form Mia-CK [14]. The cytosolic and the mitochondrial isoforms share 60-65% amino acid identity, whereas the Mi a- and Mi~-CKs share 82-85% identitx5 The similarity between the CKs and arginine kinases is lower, with -32-38% amino acid identity [15]. Our review focuses on the structure of Mi-CK [16"], as well as the mutagenesis [17",18"] and small-angle scattering [7"] studies published during the past year, and discusses possible functional implications [19",20"]. Crystal structure
The structure of Mib-CK has been solved at 3/~, resolution in space group P42 using X-ray crystallography [16"]. Analysis of the diffraction intensity data by the rotation function revealed 422 point group symmetry of the octamer and its orientation with respect to the cell axes. The unit cell contains two octamers, each at a special position, with half an octamer forming the asymmetric unit. The structure was solved to 5 ~ resolution using isomorphous replacement and to 3 ~ using phase extension, by exploiting the fourfold redundancy of the electron density. All of the four crystallographically independent monomers adopt a nearly identical structure (except for residues 1-10, 60-66, 109-120 and 313-327), consisting of a small (residues 1-112) and a large (residues 113-380) domain as shown in Figure 1. T h e small domain I comprises a short 310 helix and five ot helices, of which helix c(3 is distorted. T h e large domain II comprises an eight-stranded antiparallel I]-pleated sheet flanked by seven c~ helices, which is similar to the C-terminal domain of glutamine synthetase ([21]; A Murzin, K Fritz-Wolf, W Kabsch, unpublished data); 148 residue pairs can be superimposed with a rmsd of 3.3 A. As shown in Figure 1, the common
812
Catalysis and regulation
structure consists of a duplicated 13c~[313o~folding element which is related by an approximate twofold rotation. Moreover, both enzymes bind ATP in a similar position within the common structure, suggesting that the two enzyme families are evolutionarily related despite the absence of significant similarities in their amino acid sequences. T h e conserved amino acid residues of the CK isoenzymes (Ile52-His92, TyrlZ0-Ile132, Gln180-Lys242, Phe266His291, Arg311-Va1325, and Asp330-Va1352) form a compact cluster (shown in red in Figure 2a) in the 3D structure, covering the catalytic region of the enzyme. As expected, isoform-specific functions of Mib-CK, such as the formation of octamers or binding to the mitochondrial membrane, involve residues in the less-conserved regions of the amino acid chain.
All CK isoenzymes form stable dimers that disintegrate into monomers only under chaotropic conditions. Normally the dissociation of Mib-CK octamers upon dilution takes hours to weeks [22]; however, it takes only minutes in the presence of a transition state analogue complex (TSAC) mixture of Cr, MgADP, and nitrate [8]. Mutational studies have shown the importance of Trp206 [23] for dimer stabilit'L and Trp264 [22,23] as well as the N-terminal heptapeptide [24] for octamer stability. These observations are explained by the interactions between monomers found in the crystal structure. As shown by colour coding in Figure 2a, each monomer contains four contact regions: Contact 1 (green), LysS-Lys20 and Pro31: Contact 2 (blue and pink), Tyr34 and Ser47-Asn58; Contact 3 (violet and pink), Asn44-Gly45, Ser142-Arg147, Thr172, Lysl91 and Arg204-Trp206; and Contact 4
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Schematic representation of the secondary structure of Mi-CK. First and last amino acid residues in the ~ helices and 13-sheet strands are specified. Thick lines indicate portions of the polypeptide chain that are structurally equivalent with glutamine synthetase [21]. First and last residues of equivalent stretches are specified for Mi-CK and given in parentheses for glutamine synthetase. The single-letter amino acid code is used. The common structure consists of a 1~(~1313otfolding element and its repeat, which are related by an approximate twofold rotation as indicated by the black oval.
Mitochondrial creatine kinase Kabsch and Fritz-Wolf
(yellow), Gly134-Ser136 and Glu261-Trp264. As judged from the total energy of interaction, dimer formation involves Contacts 1-3, whereas octamer assembly involves Contacts 1 and 4. The contacts mainly involve isotypespecific regions in the amino acid sequence outside the conserved CK framework. Exceptions are Ile52-Asn58 in Contact 2 and residues Lysl91 and Arg204-Trp206 in Contact 3, which are also conserved residues (shown in pink in Figure 2a), but these residues are involved in the formation of CK d i m e r s - - a common property of both the mitochondrial and cytosolic CK isoforms. The dimers derived from the crystal structure are elongated, with a banana-like shape (length 92/~, width 42/~) as shown in Figure 2c. They are oriented with their long axis roughly parallel to the fourfold axis of the octamer (Figure 2d). The form and size of the octamer, as well as those of the dimers and their orientation within the octamer, agree well with electron microscopic results [25-27]. As shown in Figure 2e and 2f, the octamer fits into a tetragonal cell of dimensions 93 x 93 x 86 A, which gives it the appearance of a square protein when viewed along its fourfold axis. A channel of diameter 20 ~ extends along this axis through the entire octamer (Figure 2£). The N termini of the monomers protrude into the channel half way up, whereas the C termini are located at the top and bottom fourfold faces (octamer faces are perpendicular to the fourfold axis). Active site and catalytic mechanism
The analysis of ATP-containing cocrystals of Mia-CK grown in the absence of My2+ reveals a protein structure rather similar to that of the native enzyme. One ATP molecule is bound to each monomer [16"'], but each ATP molecule is found at a somewhat different place with respect to its protein environment. Moreover, the solvent exposure of the phosphate moieties, in addition to the high temperature factors for all nucleotides and their surrounding loops, indicates that none of them has reached the correct location for catalysis. As shown by small-angle scattering, the addition of ATP or ADP alone does not change the scattering curves, whereas a strong decrease of the radius of gyration of Mih-CK occurs upon binding of MgATP or TSAC [7"']. Unfortunately, crystallographic data of Mib-CK in the presence of My2+ or TSAC are not available to show the details of these large conformational changes, and only indirect evidence exists for which parts of the structure might be involved. As shown in Figure 2b, rearrangements of the structure to shield the active site from water possibly involve residues 59-66, 171-210 and 316-326. The loops 59-66, 185-196 and 316-326 are flexible, as indicated by high temperature factors and different conformations assumed in the crystallographically independent monomers, and contain the hydrophobic residues Phe63, I1e64, I1e183, Leu 188, Va1193, and Va1320 which are exposed to solvent. Moreover, Ala323 has been identified as a cleavage site for proteases and was thought to be located in an exposed surface loop involved in substrate-induced conformational
813
changes that are essential for catalysis [28]. Mutations of His61, which is found -17/~. away from ATP in the crystal structure, were shown to have a strong negative effect on the enzyme activity [17"], supporting the idea that the loop 59-66 might move towards the active site during catalysis. Although the ATP molecules found in the structure are not in their correct places required for catalysis, they cannot be far off. T h e y are located in the cleft between the domains in the highly conserved region of all guanidino kinases and many of the active-site residues implicated by various other methods, namely Cys278, Va1232-Lys237, Va1275-Arg287, Asp335, Trp223 and Ala323 are found in the vicinity of the nucleotides [23,28-32]. Details of the ATP environment are shown in Figure 3. The adenine base fits into a pocket formed by residues Met235, His186, Ser123, His291, Gly289, Arg125, and the pair ART287, Asp330 that forms a salt bridge. No specific interactions take place between these amino acids and the adenine base, whereas the phosphate groups interact with five arginine residues at positions 287, 125, 127, 315, and 336. An additional residue, ART91, is found at a distance of 5.4~ from the y-phosphate. The Cr-binding site is expected to be near Cys278 (Cys283 in cytosolic CKs), as shown by affinity labelling of this residue with epoxycreatine [29]. In the X-ray structure, Cys278 is located near the y-phosphate of ATP and Asp228, Glu227, and Glu226 (see lower right, Figure 3b), which are possible candidates for the binding of both Cr and PCr, as well as for coordinating the MgZ+ ion of ATE Cys278 was long thought to be directly involved in catalysis because its modification by sulfhydryl blocking reagents usually leads to loss of enzymatic activity [1,2], and its location in the X-ray structure seems to be compatible with this role. Kinetic studies of several replacement mutants have shown, however, that Cys278 is not essential for catalysis but is important for the synergism of substrate binding [30]. NMR studies have revealed four histidine residues located at distances of 12, 12, 14 and >18~ from the Cr 3+ in the paramagnetic, T-bidentate Cr3+ATP complex bound to the enzyme [33], and one of these histidines, with pKa =7, has been postulated to act as an acid/base catalyst [34]. In the X-ray structure His92, His291, His229, His61, and His186 are found at distances of 12, 12, 14, 17 and 18~ from the O7 atom of ATE respectively (Figure 3b). His92, which corresponds to the conserved Hisl01 of the arginine kinases [15], is found closest to the y-phosphate of ATP and to Cys278. Recent studies with replacement mutants have shown, however, that none of the conserved histidines can be considered essential for enzyme function [18°]. The largest reduction of the enzyme activity has been found for the mutant His61--)Ala, although this residue is not conserved within the enzyme family [17°]. The study was motivated by the hypothesis derived from the X-ray structure that His61 resides on the flexible loop
814
Catalysisand regulation
Figure 2
Monomer structure and octamer assembly of Mib-CK. (a) Ribbon plot [39] representing one monomer with bound ATP. The ATP is coloured in yellow, red and blue according to atom type. The common core (CC; red) of conserved amino acid residues of CK isoenzymes includes the catalytic centre of the molecule. Assembly into octamers involves four contact regions in each monomer. Contact 1 : Lys5-Lys20 and Pro31 (green). Contact 2:Tyr34 and Ser47-Cys51 (blue); Ile52-Asn58 (pink). Contact 3: Asn44-Gly45, Ser142-Arg147 and Thr172 (violet); Lys191 and Arg204-Trp206 (pink). Contact 4: Gly134-Ser136, Glu261-Trp264 (yellow). The pink regions in Contacts 2 and 3 are also part of the conserved region. The region M, Lys360-Lys380 (cyan), is involved in binding to the negatively charged headgroups of lipids in the mitochondrial membranes. (b) Hypothetical monomer structure in the presence of Mg2+ATP and Cr (shown in yellow, red and blue). The expected rearrangements of the structure upon substrate binding involve ATP and the regions 59-66, 171-210 and 316-326 (yellow). Hypothetical binding positions for Mg 2+ (shown as a sphere) in the neighbourhood of residues Asp228, Glu227 and Glu226 (blue), and for Cr near 0ys278, are indicated. Possible access routes to the active site are marked by arrows. (c) Dimer formation. Two monomers (red and pink) assemble into elongated dimers. The position of Trp206 in Contact 3 is indicated. (d) Assembly of two dimers. The position of Trp264, a residue which is known to contribute to the stability of the octamer by contacting an N-terminal Contact 1 (green) of a neighbouring dimer, is indicated. (e) Assembly of the Mib-CK octamer from four dimers. The viewing direction is roughly along a twofold axis of the octamer. (f) View of the Mib-CK octamer along its fourfold symmetry axis. A channel of 20A diameter extends through the whole octamer. In figures c-f ATP is shown in blue. Adapted with permission from [16°°].
59-66, which could move towards the active site during catalysis (see above).
As pointed out by Stroud [19 °] and Kenyon [20"], the negative results obtained from these mutational studies,
Mitochondrial creatine kinase Kabsch and Fritz-Wolf
815
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The active site of Mib-CK. (a) Schematic drawing of ATP and surrounding amino acid residues. Distances between atoms are given in hngstr6ms. The dashed lines represent possible hydrogen bonds. (b) Stereodiagram of all atoms in the environment of ATP. Single-letter amino acid code is used. Thick lines denote main-chain atoms whereas thin lines denote side-chain atoms. Adapted with permission from [16"].
which are designed to identify catalytic residues, suggest that CK might belong to the category of'conzymes', whose function is to align the substrates and to provide the appropriate electrostatic field for charge stabilisation in the transition state. T h e abundance of arginines, in particular Arg91, found in the environment of ATP are well suited to play parts in this role.
M e m b r a n e binding
T h e octamers have been shown to preferentially bind with their fourfold faces to the negatively charged phospholipid headgroups of inner and outer mitochondrial membranes, mainly by electrostatic interaction [26,35]. T h e C-terminal region Lys360-Lys380 (shown in cyan and denoted M in Figure 2a), which is located at the fourfold faces of
816
Catalysis and regulation
the octamer, indeed contains several positively charged amino acids. Some of the residues--Lys360, Lys361, Lys364, Lys369, Arg379 and Lys380, and possibly also His 113 and Lys 110--are probably involved in the binding of Mia-CK octamers to naturally occurring cardiolipin of the mitochondrial inner membrane and to the negatively charged headgroups of phospholipids found in the outer membrane. The putative membrane-binding regions at the top and bottom fourfold faces are specific to Mi-CK sequences and could explain the unique ability of Mib-CK octamets to mediate the observed adhesion between inner and outer mitochondtial membranes [35]. Moreover, the central 2 0 ~ wide channel through the octamer would then be less accessible to the surrounding intermembrane space which could be of significance for a 'back door' mechanism of Mib-CK proposed recently [36"]. In this model, ATE ADE and Cr access the active site from the intermembrane space, whereas PCr is released via an alternative route into the central channel to be exported into the cytosol via porin (Figure 4). Modelling the expected movement of the flexible loops during catalysis in a qualitative way as described above, such alternative routes do indeed show up (Figure 2b). The continuous withdrawal of PCr from the active-site environment by the proposed back door mechanism of Mib-CK would be of great thermodynamic advantage and would strongly favour the reaction for PCr production, but, in the absence of experimental proof, it remains highly speculative.
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Current Opinion in Structural Biolog~
A model of Mi-CK interactions with porins and adenine nucleotide translocators of the mitochondrial membranes. The adenine nucleotide translocator (ANT) of the inner membrane (IM) exports ATP into the intermembrane space (IMS) and imports ADP. A r P enters the active sites of Mi-CK from IMS, whereas PCr is released via a different route into the octamer channel to be exported into the cytosol via porin located in the outer membrane (OM) above the channel.
Mi-CK is the only isoform in the CK family that forms octamers, and its structure supports the hypothesis that Mi-CK has evolved to serve an additional function as a structural protein that mediates the adhesion between inner and outer mitochondrial membranes via electrostatic interactions located at its fourfold faces. Moreover, Mi-CK octamers ate functionally and perhaPs also physically coupled to ATP/ADP translocators of the inner mitochondrial membrane, and the 20~ wide channel seen in the structure may well be of significance for the exchange of energy metabolites between the mitochondrial and cytosolic compartments if the enzyme adopts a back door mechanism. Since its discovery by Lohmann I37] in 1934 in our institute, CK has been intensively studied, and now many of the results can be explained by the structure. As usual, new questions have arisen, and some difficult old ones concerning its kinetic mechanism and catalytic residues have not yet been answered. Attempts to grow crystals of Mib-CK in the presence of MgATP or as a TSAC have not been successful so f a r - - p e r h a p s because of the instability of the octamer caused by domain movements upon binding of MgATE However, the Mia-CK structure has already helped to solve the related structures of cytosolic isoforms of CK (JKM Rao, A Wlodawer, personal communication), lobster arginine kinase (C Dumas, J Janin, personal communication) and horseshoe crab arginine kinase (MS Chapman, personal communication). These high-resolution structures are expected to contribute a wealth of new information and, hopefully, will soon show the first TSAC of a guanidino kinase [38"]. Meanwhile, a structural similarity between the C-terminal domains of Mib-CK and glutamine synthetase has been found which comprises 148 residue pairs that can be superimposed with a rmsd of 3.3/~,. T h e common structure, consisting of a duplicated 13otl3[3o~ folding element related by an approximate twofold rotation, and the similar binding position of ATP in both molecules could indicate that glutamine synthetase and the family of guanidino kinases shared a common ancestor before the divergence of animals and plants.
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Matrix Space
Conclusions
References
and
recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: • °*
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Mitochondrial creatine kinase Kabsch and Fritz-Wolf
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5.
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18.
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19. Stroud RM: Balancing ATP in the cell. Nat Struct Biol 1996, • 3:567-569. Interesting views of the role and enzymatic mechanism of creatine kinase are presented. 20. Kenyon GL: Creatine kinase shapes up. Nature 1996, • 381:281-282. interesting views and comments are presented concerning the mitochondrial creatine kinase structure and the role of Cys 278 and histidines in the enzymatic mechanism. 21.
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22.
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23.
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Mitochondrial creatine k i n a s e - a square protein Wolfgang Kabsch* and Karin Fritz-Wolf The recently determined structure of octameric mitochondrial creatine kinase has provided new insights into the functioning of this enzyme and its role in channelling energy from the mitochondria to the cytoplasm. Creatine kinase, a member of the family of guanidino kinases, is structurally similar to glutamine synthetase, suggesting a possible evolutionary link between both protein families.
Addresses Max-Planck-lnstitut for medizinische Forschung, Abteilung Biophysik, Jahnstrasse 29, D-69028 Heidelberg, Germany *e-mail: [email protected] Current Opinion in Structural Biology 1997, 7:811-818
Mi-CKs are found exclusively in the mitochondrial intermembrane compartment, being attached to the inner membrane, but only the octameric form can interact with both the inner and outer rnitochondrial membranes [9"]. Mi-CK is functionally and perhaps also physically coupled to the adenine nucleotide translocator [10"'] of the inner membrane and to porin [11[ of the outer membrane. This enables the enzyme to utilise the intramitochondrially produced ATP for synthesis of PCr, which is exported into the cytosol for regeneration of ATP by the cytosolic isoforms. This interplay between the cytosolic CKs and the Mi-CKs, with the easily diffusible (P)Cr serving as an energy shuttle system between the cellular compartments, is referred to as the 'PCr circuit' [3,12,13].
http://biomednet.com/elecref/0959440X00700811 © Current Biology Ltd ISSN 0959-440X Abbreviations CK creatine kinase Cr creatine Mi-CK mitochondrial CK Mib-OK mitochondrial basic CK Per phosphocreatine rmsd root mean square deviation TSAC transition state analogue complex
Introduction Excitable cells and tissues such as muscle, brain, or spermatozoa consume large amounts of energy in a fluctuating manner. During activation, the required energy, in the form of ATP, is continuously and efficiently regenerated from a large pool of phosphocreatine (PCr) by the reaction PCr2-+ MgADP-+ H+-+MgATPZ-+ Cr catalysed by the enzyme creatine kinase (CK; EC 2.7.3.2; for reviews sec [1-3]). The reaction, believed to occur by a direct inline transfer of the phosphoryl group between the bound substrates [2,4,5], is accompanied by a conformational change of the enzyme [1,6,7"]. In addition to productive complexes, CK also forms a stable dead-end inhibition complex with nitrate, MgADE and Cr which mimics the transition state of the enzyme [8]. CK exists in tissue-specific (M, muscle-type; B, braintype), as well as in compartment-specific (cytosolic and mitochondrial), isoforms. T h e cytosolic forms are coupled to glycolysis to produce PCr, or are specifically associated at sites of high-energy consumption to produce ATE or balance the PCr/Cr and ATP/ADP pools in the cytosol. In contrast to the cytosolic forms, which are always homo- or heterodimeric (MM-, BB- or MB-CK), the mitochondrial creatine kinases (Mi-CKs) also form octamers comprising four dimers as stable building blocks. T h e mitochondrial isoforms are functionally coupled to oxidative phosphorylation to produce PCr.
Mi-CKs are also expressed in a tissue-specific manner and occur in two isoforms: a striated muscle-specific, 'sarcomeric' Mia-CK (b standing for basic); and the so-called 'ubiquitous', more acidic (a) form Mia-CK [14]. The cytosolic and the mitochondrial isoforms share 60-65% amino acid identity, whereas the Mi a- and Mi~-CKs share 82-85% identitx5 The similarity between the CKs and arginine kinases is lower, with -32-38% amino acid identity [15]. Our review focuses on the structure of Mi-CK [16"], as well as the mutagenesis [17",18"] and small-angle scattering [7"] studies published during the past year, and discusses possible functional implications [19",20"]. Crystal structure
The structure of Mib-CK has been solved at 3/~, resolution in space group P42 using X-ray crystallography [16"]. Analysis of the diffraction intensity data by the rotation function revealed 422 point group symmetry of the octamer and its orientation with respect to the cell axes. The unit cell contains two octamers, each at a special position, with half an octamer forming the asymmetric unit. The structure was solved to 5 ~ resolution using isomorphous replacement and to 3 ~ using phase extension, by exploiting the fourfold redundancy of the electron density. All of the four crystallographically independent monomers adopt a nearly identical structure (except for residues 1-10, 60-66, 109-120 and 313-327), consisting of a small (residues 1-112) and a large (residues 113-380) domain as shown in Figure 1. T h e small domain I comprises a short 310 helix and five ot helices, of which helix c(3 is distorted. T h e large domain II comprises an eight-stranded antiparallel I]-pleated sheet flanked by seven c~ helices, which is similar to the C-terminal domain of glutamine synthetase ([21]; A Murzin, K Fritz-Wolf, W Kabsch, unpublished data); 148 residue pairs can be superimposed with a rmsd of 3.3 A. As shown in Figure 1, the common
812
Catalysis and regulation
structure consists of a duplicated 13c~[313o~folding element which is related by an approximate twofold rotation. Moreover, both enzymes bind ATP in a similar position within the common structure, suggesting that the two enzyme families are evolutionarily related despite the absence of significant similarities in their amino acid sequences. T h e conserved amino acid residues of the CK isoenzymes (Ile52-His92, TyrlZ0-Ile132, Gln180-Lys242, Phe266His291, Arg311-Va1325, and Asp330-Va1352) form a compact cluster (shown in red in Figure 2a) in the 3D structure, covering the catalytic region of the enzyme. As expected, isoform-specific functions of Mib-CK, such as the formation of octamers or binding to the mitochondrial membrane, involve residues in the less-conserved regions of the amino acid chain.
All CK isoenzymes form stable dimers that disintegrate into monomers only under chaotropic conditions. Normally the dissociation of Mib-CK octamers upon dilution takes hours to weeks [22]; however, it takes only minutes in the presence of a transition state analogue complex (TSAC) mixture of Cr, MgADP, and nitrate [8]. Mutational studies have shown the importance of Trp206 [23] for dimer stabilit'L and Trp264 [22,23] as well as the N-terminal heptapeptide [24] for octamer stability. These observations are explained by the interactions between monomers found in the crystal structure. As shown by colour coding in Figure 2a, each monomer contains four contact regions: Contact 1 (green), LysS-Lys20 and Pro31: Contact 2 (blue and pink), Tyr34 and Ser47-Asn58; Contact 3 (violet and pink), Asn44-Gly45, Ser142-Arg147, Thr172, Lysl91 and Arg204-Trp206; and Contact 4
Figure 1
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Schematic representation of the secondary structure of Mi-CK. First and last amino acid residues in the ~ helices and 13-sheet strands are specified. Thick lines indicate portions of the polypeptide chain that are structurally equivalent with glutamine synthetase [21]. First and last residues of equivalent stretches are specified for Mi-CK and given in parentheses for glutamine synthetase. The single-letter amino acid code is used. The common structure consists of a 1~(~1313otfolding element and its repeat, which are related by an approximate twofold rotation as indicated by the black oval.
Mitochondrial creatine kinase Kabsch and Fritz-Wolf
(yellow), Gly134-Ser136 and Glu261-Trp264. As judged from the total energy of interaction, dimer formation involves Contacts 1-3, whereas octamer assembly involves Contacts 1 and 4. The contacts mainly involve isotypespecific regions in the amino acid sequence outside the conserved CK framework. Exceptions are Ile52-Asn58 in Contact 2 and residues Lysl91 and Arg204-Trp206 in Contact 3, which are also conserved residues (shown in pink in Figure 2a), but these residues are involved in the formation of CK d i m e r s - - a common property of both the mitochondrial and cytosolic CK isoforms. The dimers derived from the crystal structure are elongated, with a banana-like shape (length 92/~, width 42/~) as shown in Figure 2c. They are oriented with their long axis roughly parallel to the fourfold axis of the octamer (Figure 2d). The form and size of the octamer, as well as those of the dimers and their orientation within the octamer, agree well with electron microscopic results [25-27]. As shown in Figure 2e and 2f, the octamer fits into a tetragonal cell of dimensions 93 x 93 x 86 A, which gives it the appearance of a square protein when viewed along its fourfold axis. A channel of diameter 20 ~ extends along this axis through the entire octamer (Figure 2£). The N termini of the monomers protrude into the channel half way up, whereas the C termini are located at the top and bottom fourfold faces (octamer faces are perpendicular to the fourfold axis). Active site and catalytic mechanism
The analysis of ATP-containing cocrystals of Mia-CK grown in the absence of My2+ reveals a protein structure rather similar to that of the native enzyme. One ATP molecule is bound to each monomer [16"'], but each ATP molecule is found at a somewhat different place with respect to its protein environment. Moreover, the solvent exposure of the phosphate moieties, in addition to the high temperature factors for all nucleotides and their surrounding loops, indicates that none of them has reached the correct location for catalysis. As shown by small-angle scattering, the addition of ATP or ADP alone does not change the scattering curves, whereas a strong decrease of the radius of gyration of Mih-CK occurs upon binding of MgATP or TSAC [7"']. Unfortunately, crystallographic data of Mib-CK in the presence of My2+ or TSAC are not available to show the details of these large conformational changes, and only indirect evidence exists for which parts of the structure might be involved. As shown in Figure 2b, rearrangements of the structure to shield the active site from water possibly involve residues 59-66, 171-210 and 316-326. The loops 59-66, 185-196 and 316-326 are flexible, as indicated by high temperature factors and different conformations assumed in the crystallographically independent monomers, and contain the hydrophobic residues Phe63, I1e64, I1e183, Leu 188, Va1193, and Va1320 which are exposed to solvent. Moreover, Ala323 has been identified as a cleavage site for proteases and was thought to be located in an exposed surface loop involved in substrate-induced conformational
813
changes that are essential for catalysis [28]. Mutations of His61, which is found -17/~. away from ATP in the crystal structure, were shown to have a strong negative effect on the enzyme activity [17"], supporting the idea that the loop 59-66 might move towards the active site during catalysis. Although the ATP molecules found in the structure are not in their correct places required for catalysis, they cannot be far off. T h e y are located in the cleft between the domains in the highly conserved region of all guanidino kinases and many of the active-site residues implicated by various other methods, namely Cys278, Va1232-Lys237, Va1275-Arg287, Asp335, Trp223 and Ala323 are found in the vicinity of the nucleotides [23,28-32]. Details of the ATP environment are shown in Figure 3. The adenine base fits into a pocket formed by residues Met235, His186, Ser123, His291, Gly289, Arg125, and the pair ART287, Asp330 that forms a salt bridge. No specific interactions take place between these amino acids and the adenine base, whereas the phosphate groups interact with five arginine residues at positions 287, 125, 127, 315, and 336. An additional residue, ART91, is found at a distance of 5.4~ from the y-phosphate. The Cr-binding site is expected to be near Cys278 (Cys283 in cytosolic CKs), as shown by affinity labelling of this residue with epoxycreatine [29]. In the X-ray structure, Cys278 is located near the y-phosphate of ATP and Asp228, Glu227, and Glu226 (see lower right, Figure 3b), which are possible candidates for the binding of both Cr and PCr, as well as for coordinating the MgZ+ ion of ATE Cys278 was long thought to be directly involved in catalysis because its modification by sulfhydryl blocking reagents usually leads to loss of enzymatic activity [1,2], and its location in the X-ray structure seems to be compatible with this role. Kinetic studies of several replacement mutants have shown, however, that Cys278 is not essential for catalysis but is important for the synergism of substrate binding [30]. NMR studies have revealed four histidine residues located at distances of 12, 12, 14 and >18~ from the Cr 3+ in the paramagnetic, T-bidentate Cr3+ATP complex bound to the enzyme [33], and one of these histidines, with pKa =7, has been postulated to act as an acid/base catalyst [34]. In the X-ray structure His92, His291, His229, His61, and His186 are found at distances of 12, 12, 14, 17 and 18~ from the O7 atom of ATE respectively (Figure 3b). His92, which corresponds to the conserved Hisl01 of the arginine kinases [15], is found closest to the y-phosphate of ATP and to Cys278. Recent studies with replacement mutants have shown, however, that none of the conserved histidines can be considered essential for enzyme function [18°]. The largest reduction of the enzyme activity has been found for the mutant His61--)Ala, although this residue is not conserved within the enzyme family [17°]. The study was motivated by the hypothesis derived from the X-ray structure that His61 resides on the flexible loop
814
Catalysisand regulation
Figure 2
Monomer structure and octamer assembly of Mib-CK. (a) Ribbon plot [39] representing one monomer with bound ATP. The ATP is coloured in yellow, red and blue according to atom type. The common core (CC; red) of conserved amino acid residues of CK isoenzymes includes the catalytic centre of the molecule. Assembly into octamers involves four contact regions in each monomer. Contact 1 : Lys5-Lys20 and Pro31 (green). Contact 2:Tyr34 and Ser47-Cys51 (blue); Ile52-Asn58 (pink). Contact 3: Asn44-Gly45, Ser142-Arg147 and Thr172 (violet); Lys191 and Arg204-Trp206 (pink). Contact 4: Gly134-Ser136, Glu261-Trp264 (yellow). The pink regions in Contacts 2 and 3 are also part of the conserved region. The region M, Lys360-Lys380 (cyan), is involved in binding to the negatively charged headgroups of lipids in the mitochondrial membranes. (b) Hypothetical monomer structure in the presence of Mg2+ATP and Cr (shown in yellow, red and blue). The expected rearrangements of the structure upon substrate binding involve ATP and the regions 59-66, 171-210 and 316-326 (yellow). Hypothetical binding positions for Mg 2+ (shown as a sphere) in the neighbourhood of residues Asp228, Glu227 and Glu226 (blue), and for Cr near 0ys278, are indicated. Possible access routes to the active site are marked by arrows. (c) Dimer formation. Two monomers (red and pink) assemble into elongated dimers. The position of Trp206 in Contact 3 is indicated. (d) Assembly of two dimers. The position of Trp264, a residue which is known to contribute to the stability of the octamer by contacting an N-terminal Contact 1 (green) of a neighbouring dimer, is indicated. (e) Assembly of the Mib-CK octamer from four dimers. The viewing direction is roughly along a twofold axis of the octamer. (f) View of the Mib-CK octamer along its fourfold symmetry axis. A channel of 20A diameter extends through the whole octamer. In figures c-f ATP is shown in blue. Adapted with permission from [16°°].
59-66, which could move towards the active site during catalysis (see above).
As pointed out by Stroud [19 °] and Kenyon [20"], the negative results obtained from these mutational studies,
Mitochondrial creatine kinase Kabsch and Fritz-Wolf
815
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Current Opinion in Structurat Biology
The active site of Mib-CK. (a) Schematic drawing of ATP and surrounding amino acid residues. Distances between atoms are given in hngstr6ms. The dashed lines represent possible hydrogen bonds. (b) Stereodiagram of all atoms in the environment of ATP. Single-letter amino acid code is used. Thick lines denote main-chain atoms whereas thin lines denote side-chain atoms. Adapted with permission from [16"].
which are designed to identify catalytic residues, suggest that CK might belong to the category of'conzymes', whose function is to align the substrates and to provide the appropriate electrostatic field for charge stabilisation in the transition state. T h e abundance of arginines, in particular Arg91, found in the environment of ATP are well suited to play parts in this role.
M e m b r a n e binding
T h e octamers have been shown to preferentially bind with their fourfold faces to the negatively charged phospholipid headgroups of inner and outer mitochondrial membranes, mainly by electrostatic interaction [26,35]. T h e C-terminal region Lys360-Lys380 (shown in cyan and denoted M in Figure 2a), which is located at the fourfold faces of
816
Catalysis and regulation
the octamer, indeed contains several positively charged amino acids. Some of the residues--Lys360, Lys361, Lys364, Lys369, Arg379 and Lys380, and possibly also His 113 and Lys 110--are probably involved in the binding of Mia-CK octamers to naturally occurring cardiolipin of the mitochondrial inner membrane and to the negatively charged headgroups of phospholipids found in the outer membrane. The putative membrane-binding regions at the top and bottom fourfold faces are specific to Mi-CK sequences and could explain the unique ability of Mib-CK octamets to mediate the observed adhesion between inner and outer mitochondtial membranes [35]. Moreover, the central 2 0 ~ wide channel through the octamer would then be less accessible to the surrounding intermembrane space which could be of significance for a 'back door' mechanism of Mib-CK proposed recently [36"]. In this model, ATE ADE and Cr access the active site from the intermembrane space, whereas PCr is released via an alternative route into the central channel to be exported into the cytosol via porin (Figure 4). Modelling the expected movement of the flexible loops during catalysis in a qualitative way as described above, such alternative routes do indeed show up (Figure 2b). The continuous withdrawal of PCr from the active-site environment by the proposed back door mechanism of Mib-CK would be of great thermodynamic advantage and would strongly favour the reaction for PCr production, but, in the absence of experimental proof, it remains highly speculative.
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Current Opinion in Structural Biolog~
A model of Mi-CK interactions with porins and adenine nucleotide translocators of the mitochondrial membranes. The adenine nucleotide translocator (ANT) of the inner membrane (IM) exports ATP into the intermembrane space (IMS) and imports ADP. A r P enters the active sites of Mi-CK from IMS, whereas PCr is released via a different route into the octamer channel to be exported into the cytosol via porin located in the outer membrane (OM) above the channel.
Mi-CK is the only isoform in the CK family that forms octamers, and its structure supports the hypothesis that Mi-CK has evolved to serve an additional function as a structural protein that mediates the adhesion between inner and outer mitochondrial membranes via electrostatic interactions located at its fourfold faces. Moreover, Mi-CK octamers ate functionally and perhaPs also physically coupled to ATP/ADP translocators of the inner mitochondrial membrane, and the 20~ wide channel seen in the structure may well be of significance for the exchange of energy metabolites between the mitochondrial and cytosolic compartments if the enzyme adopts a back door mechanism. Since its discovery by Lohmann I37] in 1934 in our institute, CK has been intensively studied, and now many of the results can be explained by the structure. As usual, new questions have arisen, and some difficult old ones concerning its kinetic mechanism and catalytic residues have not yet been answered. Attempts to grow crystals of Mib-CK in the presence of MgATP or as a TSAC have not been successful so f a r - - p e r h a p s because of the instability of the octamer caused by domain movements upon binding of MgATE However, the Mia-CK structure has already helped to solve the related structures of cytosolic isoforms of CK (JKM Rao, A Wlodawer, personal communication), lobster arginine kinase (C Dumas, J Janin, personal communication) and horseshoe crab arginine kinase (MS Chapman, personal communication). These high-resolution structures are expected to contribute a wealth of new information and, hopefully, will soon show the first TSAC of a guanidino kinase [38"]. Meanwhile, a structural similarity between the C-terminal domains of Mib-CK and glutamine synthetase has been found which comprises 148 residue pairs that can be superimposed with a rmsd of 3.3/~,. T h e common structure, consisting of a duplicated 13otl3[3o~ folding element related by an approximate twofold rotation, and the similar binding position of ATP in both molecules could indicate that glutamine synthetase and the family of guanidino kinases shared a common ancestor before the divergence of animals and plants.
OM
Matrix Space
Conclusions
References
and
recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: • °*
of special interest of outstanding interest Watts DC: Creatine kinase. In The Enzymes, vol 8A, edn 3. Edited by Boyer PD. New York/London: Academic Press: 1973:383-455.
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Mitochondrial creatine kinase Kabsch and Fritz-Wolf
3.
Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM: Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis. Biochem J 1992, 281:21-40.
4.
Hansen DE, Knowles JR: The stereochemical course of the reaction catalysed by creatine kinase. J Biol Chem 1981, 256:5967-5969.
5.
Leyh TS, Goodhart PJ, Nguyen AC, Kenyon GL, Reed GH: Structures of manganese(ll) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands. Biochemistry 1985, 24:308-316.
6.
McLaughlin AC, Leigh JS, Cohn M: Magnetic resonance study of the three-dimensional structure of creatine kinase-substrate complexes. J Biol Chem 1976, 251:2777-2787.
7. •°
Forstner M, Kriechbaum M, Laggner P, Wallimann T: Changes of creatine kinase structure upon ligand binding as seen by small-angle scattering. J Mol Struct 1996, 383:217-222. This paper reports a large reduction of the radius of gyration of mitochondrial creatine kinase upon binding of MgATP or forming a transition state analog complex.
817
18.
Chen LH, Borders CL, V&squez JR, Kenyon GL: Rabbit muscle creatine kinase: consequences of the mutagenesis of conserved histidine residues. Biochemistry 1996, 35:7895-7902. The histidine residues of rabbit muscle creatine kinase, corresponding to residues 92, 101, 186, 229, 291 in chicken mitochondrial creatine kinase, are shown not to be of importance in the transphosphorylation reaction catalysed by creatine kinase. •
19. Stroud RM: Balancing ATP in the cell. Nat Struct Biol 1996, • 3:567-569. Interesting views of the role and enzymatic mechanism of creatine kinase are presented. 20. Kenyon GL: Creatine kinase shapes up. Nature 1996, • 381:281-282. interesting views and comments are presented concerning the mitochondrial creatine kinase structure and the role of Cys 278 and histidines in the enzymatic mechanism. 21.
Liaw SH, Jun G, Eisenberg D: Interactions of nucleotides with fully unadenylylated glutamine synthetase from Salmonella typhimurium. Biochemistry 1994, 33:11184-11188.
22.
Gross M, Wallimann T: Dimer-dimer interactions in octameric mitochondrial creatine kinase. Biochemistry 1995, 34:6660-6667.
Milner-White EJ, Watts DC: Inhibition of adenosine 5'-triphosphate-creatine phosphotransferase by substrate-anion complexes. Evidence for the transition-state organisation of the catalytic site. Biochem J 1971, 122:727-740.
23.
Stachowiak O, Dolder M, Wallimann T: Membrane-binding and lipid vesicle cross-linking kinetics of the mitochondrial creatine kinase octamer. Biochemistry 1996, 35:15522-15528. Using model membrane vesicles containing different amounts of cardiolipin, the authors show that only octameric mitochondrial creatine kinase induces bridged vesicle-protein complexes.
Gross M, Furter-Graves EM, Wallimann T, Eppenberger HM, Furter R: The tryptophan residues of mitochondrial creatine kinase: roles of Trp223, Trp206, and Trp264 in active-site and quaternary structure formation. Protein Science 1994, 3:1058-1068.
24.
Kaldis P, Furter R, Wallimann T: The N-terminal heptapeptide of mitochondrial creatine kinase is important for octamerization. Biochemistry 1994, 33:952-959.
25.
Schnyder T, Gross H, Winkler H, Eppenberger HM, Wallimann T: Structure of the rnitochondrial creatine kinase octamer: high resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions. J Cell Biol 1991, 112:95-101.
26.
Schnyder T, Cyrklaff M, Fuchs K, Wallimann T: Crystallization of mitochondrial creatine kinase on negatively charged lipid bilayers. J Struct Bio11994, 112:136-147.
27.
Schnyder T, Engel A, Lustig A, Wallimann T: Native mitochondrial creatine kinase (Mi-CK) forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurement by STEM and image analysis of single Mi-CK octamers. J Bio/Chem 1988, 263:16954-16962.
28.
Wyss M, James P, Schlegel J, Wallimann T: Limited proteolysis of creatine kinase. Implications for three-dimensional structure and for conformational substates. Biochemistry 1993, 32:10727-10735.
8.
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10. •.
Beutner G, ROck A, Riede B, Welte W, Brdiczka D: Complexes between kinases, mitochondrial porin and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett 1996, 396:189-195. This paper shows that hexokinase isozyme I, as well as mitochondrial creatine kinase, exists physiologically as a complex with porin and the adenine nucleotide translocator. 11.
Brdiczka D, Kaldis P, Wallimann T: In vitro complex formation between the octamer of mitochondrial creatine kinase and porin. J Biol Chem 1994, 269:27640-27644.
12.
Bessman SP, Geiger PJ: Transport of energy in muscle: the phosphorylcreatine shuttle. Science 1981, 211:449-452.
13.
Saks VA, Khuchua ZA, Vasilyeva EV, Belikova OY, Kuznetsov AV: Metabolic compartmentation and substrate channeling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration-a synthesis. Mo/Cell Biochern 1994, 133/134:155-192.
14.
Hossle JP, Schlegel J, Wegmann G, Wyss M, B6hlen P, Eppenberger HM, Wallimann T, Perriard JC: Distinct tissue specific mitochondrial creatine kinases from chicken brain and striated muscle with a conserved CK framework. Biochem Biophys Res Commun 1988, 151:408-416.
29.
Buechter DD, Medzihradszky KF, Burlingame AL, Kenyon GL: The active site of creatine kinase. Affinity labeling of cysteine 262 with epoxycreatine. J Biol Chem 1992, 267:2173-2178.
30.
M0hlebach SM, Gross M, Wirz T, Wallimann T, Perriard JC, Wyss M: Sequence homology and structure predictions of the creatine kinase isozymes. Mo/Cell Biochem 1994,
Furter R, Furter-Graves EM, Wallimann T: The reactive cysteine is required for synergism but is nonessential for catalysis. Biochemistry 1993, 32:7022-7029.
31.
Olcott MC, Bradley ML, Haley BE: Photoaffinity labeling of creatine kinase with 2-azido- and 8-azidoadenosine triphosphate: identification of two peptides from the ATPbinding domain. Biochemistry 1994, 33:11935-11941.
32.
James P, Wyss M, Lutsenko S, Wallimann T, Carafoli E: ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp 336 with ClRATP. FEBS Lett 1990, 273:139-143.
33.
Rosevear PR, Desmeules P, Kenyon GL, Mildvan AS: Nuclear magnetic resonance studies of the role of histidines at the active site of rabbit muscle creatine kinase. Biochemistry 1981, 20:6155-6164.
15.
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16. Fritz-Wolf K, Schnyder T, Wallimann T, Kabsch W: Structure of •• mitochondrial creatine kinase. Nature 1996, 381:341-345. This paper describes the first structure of a guanidino kinase. 1 7.
Forstner M, MLiller A, Stolz M, Wallimann T: The active site histidines of creatine kinase. A critical role of His61 situated on a flexible loop. Protein Sci 1997, 6:331-339. His61 of creatine kinase is shown to be important for the catalytic reaction but that it does not serve as an acid/base catalyst in the transphosphorylation of creatine and ATP. •
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34.
Cook PF, Kenyon GL, Cleland WW: Use of pH studies to elucidate the catalytic mechanism of rabbit muscle creatine kinase. Biochemistry 1981, 20:1204-1210.
35.
Rojo M, Hovius R, Demel RA, Nicolay K, Wallimann T: Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes. J Bio/Chem 1991, 266:20290-20295.
36. •
Schlattner U, Forstner M, Eder M, Stachowiak O, Fritz-Wolf K, WallimannT: Functional aspects of the X-ray structure of mitochondrial creatine kinase: a molecular physiology approach. Mo/Ceil Biochem 1997, in press. A detailed discussion of a possible functional role of the central channel and membrane-binding properties of mitochondrial creatine kinase. 37.
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der Muskelkontraktion. Biochem Z 1934, 271:264-277. [Title translation: On enzymatic hydrolysis of PCR: a contribution to chemistry of muscle contraction.] 38. •
Zhou G, Parthasarathy G, Somasundaram T, Abeles A, Roy L, Strong SJ, Ellington WR, Chapman MS: Expression, purification from inclusion bodies, and crystal characterization of a transition state analog complex of arginine kinase: a model for studying phosphagen kinases. Protein Sci 1997, 6:444-449. High-quality crystals of a transition state analog complex of horseshoe crab arginine kinase are described, which promise to yield important new insight into the enzyme mechanism. 39.
Jones TA, Zou JY, Cowan SW, Kjeldgaard M: Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr 1991, 47:110-119.