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Preliminary crystallographic analysis of human myeloperoxidase
-1. Xol. Bid. (1988) 199, 395-396
Preliminary Crystallographic Analysis of Human Myeloperoxidase Two crystal forms of human neutrophil myeloperoxidase have been grown from solutions containing polyethylene glycol as precipitant. An orthorhombic form with bipyramidal morphology has space group P4,22 or P4,22 and cell dimensions a = b = 109 A and c = 235 A. The second form is monoclinic with a hexagonal plate morphology, and has space group P2, and cell dimensions a = 112.0 A, b = 64.2 8, c = 91.5 A and /? = 97.4”. Both forms contain one 140,000 &Z, tetramer in the asymmetric unit. The monoclinic crystals diffract to a resolution better than 2.3 A and are suitable for X-ray struct,ural analysis.
Myeloperoxidase is the major haemoprotein of neutrophils and constitutes up to 5% of the weight of these cells (Schultz & Kaminker, 1962; Nauseef et al.. 1983). The enzyme is believed to have an antimicrobial function (Klebanoff, 1975). The human enzyme appears to comprise two identical carbohydrate-containing subunits of M, 57,000, and two identical carbohydrate-free subunits of M, 10,500 (Olsen & Little, 1984). Other workers have proposed alternative models for the quaternary structure of this enzyme (see for example, Andersen et al., 1982; Nauseef et al., 1983; Nauseef & Malech, 1986). Myeloperoxidase contains two tightly bound haem prosthetic groups (Agner, 1958). The enzyme is synthesized in HL-60 cells as a 75,000 to 80,000 .Vr precursor polypeptide (Olsson et al., 1984: Hasilik ct aZ., 1984). The gene sequence and t,hereby the amino acid sequence for this precursor has been published (Johnson et al., 1987). (‘rystals of myeloperoxidase were first reported by Agner (1958) and by Schultz (1958) working wlt)h the canine and rat enzyme, respectively. Harrison et al. (1977) subsequently described a different, crystallization procedure for canine myeloperoxidase. and Fenna (1987) has characterized three crystal forms of this enzyme. Crystallization of human myeloperoxidase has been reported by Morita et al. (1986), but the small crystals were not &aracterized by X-ray analysis. We have now grown much larger crystals of human myelopcroxidase and present’ the results of a preliminary X-ray crystallographic analysis. Myeloperoxidase was isolated from the buffy (boats of out’dated human blood as described by Olsen & Little (1983). For crystallization, protein solutions were obtained by twice washing by resuspension a precipitate (from 65”/;, sat’urated ammonium sulphate suspension) in 300/ (w/v) polyet,hylene glycol 6000. The final pellet/oil was dissolved in water to a concent’ration of 30 mg/ml. The enzyme solution (0.1 ml) was mixed with 0.1 ml of 20 ‘;& (w/v) polyethylene glycol 4000 or 6000, placed in a plastic well and sealed in the presence of I ml of 20yCj (w/v) polyethylene glucol 4000 or 6000
according to whichever had been added to the enzyme solution. After two or three weeks at room temperature, enzyme samples in polyethylene glycol 4000 yielded either thin hexagonal plates typically 0.8 mm x 0.4 mm, or bipyramidal crystals of up to 0.5 mm in each dimension. From solutions in polyethylene glycol 6000. thicker hexagonal plates were obtained, typically 0.8 mm x O-6 mm with a thickness of up to 0.15 mm. The bipyramidal crystals belong to the tetragonal space group P4,22 or P4,22, with cell dimensions a = b = 109.0( kO.3) A and c = 2354( *o-7) A (1 a = 0.1 nm). The asymmetric unit, czontains one tetramer (M, 140,000), and the volume to molecular weight ratio, 1, (Matthews, 1968), is 2.50 a,nd close t’o the median value observed for protein c,rystals. The crystals diffract poorly, however, t)o a resolution limit of approximately 6 x only, and changes in the a and b cell dimension (contraction lo 105.6( fO.3) a) were observed from omh cryst,al during irradiation. The hexagonal plate crystals belong t,o t’he monoclinic space group P2, with cell dimensions n = 1 12.0( + 0.3) x, b = 64.2( +0.3) A, (’ = 91.5 ( f 0.3) ,4 and /I = 97.4( f 0.2)“. The cell parameters were estimated from 15” precession photographs, and reflections corresponding t’o a resolut,ion of 2.3 A were observed on still photographs. The asymmetric unit’ of this crystal form also contains a single tetramer. and the I/;, ratio is 2.33. The density of the crystals, measured by t)hr m&hod of I,ow & Richards (1952) in a ~)romol)c~nz~~t~e/xylene density gradient. was found to b(b 1.2X( kO.01) g cm -3. This value, and the experimrnt~al value of 0.73 cm3 g-l for the partial specific volume of this protein (Ehrenberg & Agner, 1958). and assuming totally salt-free and polyethylene plyc~)l-frr~ solvent channels wit’hin the crystal, leads to an overestimate of the molecular weight, of t)hr protein by 45”b. Unexpectedly high crystal densities have been recorded for other protein crystals grown frorn polyethylene glycol (Aschaffenburg rt al.. 1979), and it has been suggested that the solvent rhannels may cont.ain low molecular weight) polymers of cathylene
glyc~l. Interestingly. the density reported hy Fenna (1985) for cryst,als of canine my~~loperoxitlas~,. grown from a solution cvntaining Stnethyl-2.4. perttjanediol. is also high and leads to an apparent overestimation of the molecular weight. The shape of the myeloprroxidase molecule has been investigated both hy ckvtron microscopy of the guinea, pig enzyme (Desser d (1~1.. 197%) and analytical ultracentnfugation of the hutnan enzyme (Xndrews $ Krinsky, 1981: Morita it al.. 1986). L!Thile the latter studies indicate axial ratios of 5 : I and 3 : 1, the fortner suggests a value nearer 1% : I. Although a molecule with t)he shape of it prolate spheroid and an axial ratio of 3 : 1 can br ac*c*otrtmodated wit,hin the t.rt ragonal unit cvll. neither of the two higher axial ratios is consistent wit)h acceptable tnolec~ular packing arrangements wit’hin t)hrl monoclinicb unit) cell. Thra modvl proposed by electron microscopy. however (2 prolate spheroids 60 !I x .50 .A x 50 A apposed at their is ettt’irely consist,ent and, smaller dimension). furthermore. two of the possible molecular packing arrangements ar(l consistent also with the wesk suggt&on of non-crystallographic. symmetry t,hat is risible in the diffraction pat’tern of the hOl projwtion. The tton-rr?-st~~tllog~~a~~hi(~ d-fold axis appears to lie approximately along or perpendicular to thv direction [ 2 0 - I] and relates the two. presumably ident’ical, halves of the molecule. crystals of human mveloThe monoclinic peroxidase reported here are c,learly suitable i(x a higbrrsolution
the search This (lS..l.S.) and thr
X-ray
c,r~stallo~r~1)hic.
for heavy-atom
work was supported and Thr Norwegian Humanities (C’.T,.).
analysis.
derivatives by Rtwarcoh
The Royal Socaicxt! (‘ounc,il f’or Scir~nw
Brian J. Sutton I)rl)art~nlrnt of ISiophysicx ( ‘ell anti King’s (‘ollrgc~ London. Z(i- “9 bury Lontlon W(*:!R ;iR,L. I‘.K.
Clive Little Ragnar L. Olsen Nils Peder Willassen
ad
is underway.
Molecular Lanr
I~iolog?
Preliminary Crystallographic Analysis of Human Myeloperoxidase Two crystal forms of human neutrophil myeloperoxidase have been grown from solutions containing polyethylene glycol as precipitant. An orthorhombic form with bipyramidal morphology has space group P4,22 or P4,22 and cell dimensions a = b = 109 A and c = 235 A. The second form is monoclinic with a hexagonal plate morphology, and has space group P2, and cell dimensions a = 112.0 A, b = 64.2 8, c = 91.5 A and /? = 97.4”. Both forms contain one 140,000 &Z, tetramer in the asymmetric unit. The monoclinic crystals diffract to a resolution better than 2.3 A and are suitable for X-ray struct,ural analysis.
Myeloperoxidase is the major haemoprotein of neutrophils and constitutes up to 5% of the weight of these cells (Schultz & Kaminker, 1962; Nauseef et al.. 1983). The enzyme is believed to have an antimicrobial function (Klebanoff, 1975). The human enzyme appears to comprise two identical carbohydrate-containing subunits of M, 57,000, and two identical carbohydrate-free subunits of M, 10,500 (Olsen & Little, 1984). Other workers have proposed alternative models for the quaternary structure of this enzyme (see for example, Andersen et al., 1982; Nauseef et al., 1983; Nauseef & Malech, 1986). Myeloperoxidase contains two tightly bound haem prosthetic groups (Agner, 1958). The enzyme is synthesized in HL-60 cells as a 75,000 to 80,000 .Vr precursor polypeptide (Olsson et al., 1984: Hasilik ct aZ., 1984). The gene sequence and t,hereby the amino acid sequence for this precursor has been published (Johnson et al., 1987). (‘rystals of myeloperoxidase were first reported by Agner (1958) and by Schultz (1958) working wlt)h the canine and rat enzyme, respectively. Harrison et al. (1977) subsequently described a different, crystallization procedure for canine myeloperoxidase. and Fenna (1987) has characterized three crystal forms of this enzyme. Crystallization of human myeloperoxidase has been reported by Morita et al. (1986), but the small crystals were not &aracterized by X-ray analysis. We have now grown much larger crystals of human myelopcroxidase and present’ the results of a preliminary X-ray crystallographic analysis. Myeloperoxidase was isolated from the buffy (boats of out’dated human blood as described by Olsen & Little (1983). For crystallization, protein solutions were obtained by twice washing by resuspension a precipitate (from 65”/;, sat’urated ammonium sulphate suspension) in 300/ (w/v) polyet,hylene glycol 6000. The final pellet/oil was dissolved in water to a concent’ration of 30 mg/ml. The enzyme solution (0.1 ml) was mixed with 0.1 ml of 20 ‘;& (w/v) polyethylene glycol 4000 or 6000, placed in a plastic well and sealed in the presence of I ml of 20yCj (w/v) polyethylene glucol 4000 or 6000
according to whichever had been added to the enzyme solution. After two or three weeks at room temperature, enzyme samples in polyethylene glycol 4000 yielded either thin hexagonal plates typically 0.8 mm x 0.4 mm, or bipyramidal crystals of up to 0.5 mm in each dimension. From solutions in polyethylene glycol 6000. thicker hexagonal plates were obtained, typically 0.8 mm x O-6 mm with a thickness of up to 0.15 mm. The bipyramidal crystals belong to the tetragonal space group P4,22 or P4,22, with cell dimensions a = b = 109.0( kO.3) A and c = 2354( *o-7) A (1 a = 0.1 nm). The asymmetric unit, czontains one tetramer (M, 140,000), and the volume to molecular weight ratio, 1, (Matthews, 1968), is 2.50 a,nd close t’o the median value observed for protein c,rystals. The crystals diffract poorly, however, t)o a resolution limit of approximately 6 x only, and changes in the a and b cell dimension (contraction lo 105.6( fO.3) a) were observed from omh cryst,al during irradiation. The hexagonal plate crystals belong t,o t’he monoclinic space group P2, with cell dimensions n = 1 12.0( + 0.3) x, b = 64.2( +0.3) A, (’ = 91.5 ( f 0.3) ,4 and /I = 97.4( f 0.2)“. The cell parameters were estimated from 15” precession photographs, and reflections corresponding t’o a resolut,ion of 2.3 A were observed on still photographs. The asymmetric unit’ of this crystal form also contains a single tetramer. and the I/;, ratio is 2.33. The density of the crystals, measured by t)hr m&hod of I,ow & Richards (1952) in a ~)romol)c~nz~~t~e/xylene density gradient. was found to b(b 1.2X( kO.01) g cm -3. This value, and the experimrnt~al value of 0.73 cm3 g-l for the partial specific volume of this protein (Ehrenberg & Agner, 1958). and assuming totally salt-free and polyethylene plyc~)l-frr~ solvent channels wit’hin the crystal, leads to an overestimate of the molecular weight, of t)hr protein by 45”b. Unexpectedly high crystal densities have been recorded for other protein crystals grown frorn polyethylene glycol (Aschaffenburg rt al.. 1979), and it has been suggested that the solvent rhannels may cont.ain low molecular weight) polymers of cathylene
glyc~l. Interestingly. the density reported hy Fenna (1985) for cryst,als of canine my~~loperoxitlas~,. grown from a solution cvntaining Stnethyl-2.4. perttjanediol. is also high and leads to an apparent overestimation of the molecular weight. The shape of the myeloprroxidase molecule has been investigated both hy ckvtron microscopy of the guinea, pig enzyme (Desser d (1~1.. 197%) and analytical ultracentnfugation of the hutnan enzyme (Xndrews $ Krinsky, 1981: Morita it al.. 1986). L!Thile the latter studies indicate axial ratios of 5 : I and 3 : 1, the fortner suggests a value nearer 1% : I. Although a molecule with t)he shape of it prolate spheroid and an axial ratio of 3 : 1 can br ac*c*otrtmodated wit,hin the t.rt ragonal unit cvll. neither of the two higher axial ratios is consistent wit)h acceptable tnolec~ular packing arrangements wit’hin t)hrl monoclinicb unit) cell. Thra modvl proposed by electron microscopy. however (2 prolate spheroids 60 !I x .50 .A x 50 A apposed at their is ettt’irely consist,ent and, smaller dimension). furthermore. two of the possible molecular packing arrangements ar(l consistent also with the wesk suggt&on of non-crystallographic. symmetry t,hat is risible in the diffraction pat’tern of the hOl projwtion. The tton-rr?-st~~tllog~~a~~hi(~ d-fold axis appears to lie approximately along or perpendicular to thv direction [ 2 0 - I] and relates the two. presumably ident’ical, halves of the molecule. crystals of human mveloThe monoclinic peroxidase reported here are c,learly suitable i(x a higbrrsolution
the search This (lS..l.S.) and thr
X-ray
c,r~stallo~r~1)hic.
for heavy-atom
work was supported and Thr Norwegian Humanities (C’.T,.).
analysis.
derivatives by Rtwarcoh
The Royal Socaicxt! (‘ounc,il f’or Scir~nw
Brian J. Sutton I)rl)art~nlrnt of ISiophysicx ( ‘ell anti King’s (‘ollrgc~ London. Z(i- “9 bury Lontlon W(*:!R ;iR,L. I‘.K.
Clive Little Ragnar L. Olsen Nils Peder Willassen
ad
is underway.
Molecular Lanr
I~iolog?