J. Mob. Rid.
(1978) 123. 285-286
Preliminary X-ray Study of Crystals of Human Transferrin
Transftarrin is responsible for iron transport to Iiclnoglo~)in-synthesizing red blood cells. Human transferrin is a glvcoprotein consisting of a single polypeptide chain of (j7G residues wit11 a molecular \\-eight of 81.000 (MacGillivray of al.. 1977). There MI’C t\vo itlcnt~ical cart)ohydrate chains with molecular weights of approximately 24(H) FL.II.+. I)oth of which are connected to asparagine on the peptidc hackbone 1)~ asparert al.. 1977). The protein is capai)k* gill~l-i\‘-;Lt~etvl-glu~osamine linkages (MacGillivrwy of binding two ferric ions tightly hut reversibly. Transferrin can also bind a varirt,! of othw metal ions. which sul)stitutta for ferric ions at thr two iron-binding sites (Putnam. 1975). Trwnsferrin is among the l)t~st-charactcrizeci of t hi mct.al t,ra.nsport proteins, in t,crrns of physical nnd hiochc~mical properties. biological function. iind int,eractions 1j.it,h tar@ wlls. Work towartl t,lrtA primar?; secptwt:t~ of human t~rwlsferrin is nearing conlJ)lt~tion (Sutton et rrl.. 1975). wd tlw cht~mical structurt~ of the t:arl)oh~dr>~tt~ chains has INYW rt~portt~tl (Spik rt ~2.. 1975). ‘Nit, rnimy ph~sicill and chtwical studies of t,r;wb(ibrrin II:\\-t, raiwd wvcraI fitIltlilttlr~nt,~~l Stl~ll~tUKll t{uwt,iolls t.llilt, might, best lNb illlS\Yl’t’l’tl l)y il cr?st;tllograpl~ic anal.ysi s of t,hr Jwot,t%l. ‘I‘htw 11;~slwtw pLt~~klJhJ intc~rt~st alld rontrovcrs~y concerning the iron-binding sitw irwluding questions al)out the t,ypt*s of amino acids that participate in t’htw sit,tbs: thta possihlr difftwntw lwtwt~t~11 the tn-o iron binding sites; the roles that anions. suc11 as hicarbonate, pIa> at thctsca sites: and the mechanisms t)y which various metal ions substitute for iron. Ttw crystal structure of transferrin should also Ilclp resolve t,he current, questions about possible internal structural homology wit’hin the molecule (MacGillivr;zy et t/l.. 1977). In addition, a high-resolution crystallographic: st’udy of t ransferrin may rtivwl thtl t~onformations of the carbohydrate moieties. which havtk bwn implicatt~d in ttw binding of transft:rrin to reticultqtc receptors (Aisw. 1!)74). (‘r~st,allograJ)hic studies of rill)t)it transftxrrin (Al-Hilal P/ (11.. 1976) irtltl of’ cllit:ktw tra~lsfrrrin (*J. E. ,1l~,I:t, Jwrsoni~.l comrnunic~~tion) arc in progress. H umall swum apotransfcrril1 11~s purch;~wd fronl Rrh ring\vtarkta C’ompany. ‘I’lrl~ nJ)ot~t~;ltlsft~l~l.irlevils corivt~rt~t~tl to transftlrrin 1)~ titrating solutions of ttltl protein in (I*-& RI-t)iwrtwnatt~ (pH 7.5) \\,ittl a11 iron complrx of‘ nit~rilotri;~t*t~t ic acid. ‘1’11thprottbin solut,ion was dia1yzt.d against a sodium Iblrbonate solut,ion t,o assurt’ rt~platwnrtrt of all rlit,t.ilot,r,i;lcetRtC anions 1)~ t)icnrt)onate. Thr solution wts t:ollct~ntr;~tt~tl t)y vacuutn dialysis against, 0#4 M-sodium nawd.ylat~r huffw (pH 5.75) t.o il prot.tbin conwntrat iwl of about 40 mg/mJ. Crpst)aJs WeJ’t’ obtained hy the batch method. using (I.3 to 0.5 nil portions of the protein solution, to which So (, polyet hylcne glvcot (average molecular wright 4000) was added. The solutions, stored at 4°C’. produced small crystals within 72 hours. Aft’er approximately ten days, red plate-like crystals with dimensions up to
286
L. J. DoLUC,zS,
F. L. SUDDATH,
R. :\. G.4318 .4ND C. I?.. BUGC,
1.5 mm x 0.5 mm x 0.4 mm were obtained. Precession photographs and diffractowith 11,: 78 a. h = 94 .f and meter data indicate that t,he cryst.als arc’ orthorhomhic. c =- 112 A. The space group is 1’2,2,2,. as indicat,ed hy bhe systematic absence of reflections hO0 with h odd, Ok0 with k odd and 001 with 2 odd. The diffraction data extend weakly to a resolution of 2.6 a on photographs taken with crystals in stationary orientations. The density of the transferrin cryst’als, as measured by the Ficoll gradient technique (Westbrook, 1976), is 1.193 g cmm3. Assuming that the unit cell parameters were unchanged in the Ficoll gradient. and that the protein has a partial specific volume of 0.725 cm3/g (Putnam. 1975). the calculated (Colman & Matthews. 1971) mass of protein per asymmetric unit is 84,884 daltons. which is not significantl! different from the molecular weight of 81.000 reported by MacGillivray (1977). Native diffractometer data to 6 p\ have been collecbed. We are currently searching for isomorphous heavy-atom derivatives. This research was supported by Unitsed States Public Healtll CA-13148, and DE-02670. Institute of Dental Research Comprehensive Cancer Centri Department of Biochemistry University of Alabama in Birmingham University Statioll, Birmingbarn Ala. 35294, U.S.A. Received 24 April
Services grants CA- 12 159,
L. J. D~LCCAS F. L. SCJDDATH K. A. (:Aiw c. E. Brrcc
1978
REFERENCES Aisen, P. (1974). B&t. J. Haematol. 26, 159--163. Al-Hilal, D., Baker, E., Carlisle, C. H., Gorinsky, B., Harsburgh, R. C., Lindley, I’. F.. Moss, D. S., Schneider, H. & Stimpson, R. (1976). J. Mol. Biol. 108, 255-257. Colman, P. M. & Matthews, B. W. (1971). J. Mol. Biol. 60, 163.-168. MacGillivray, R. T. A., Mendez, E. & Brew, K. (1977). Proteins vf Iron Metabolism (Brown, Aisen & Crichton, eds), pp. I33--142, Grunt & Stratton. New York, San Francisco, London. Putnam, E’. W. (1975). The Plasma /‘roteins (Pntnam, cd.), pp. 265 ~316. Academic Press. New York, London. Spik, U., Bayard, B., Fournet, B., Streckrr, (2.. Houquelet, S. & Montm~~il. .I. (1975). FEBS
Letters,
50, 296.-299.
Sutton, M. R., MacGillivray.
R. T. A. h Brclw, K. (1975). Proteins of lron Storage and and Il1edi&e (Crichton, ed.). pp. 19 ~26, Nort,h-Holland Publisbing Company, Amsterdam, Oxford. Westbrook, E. M. (1976). J. Mol. Viol. 103, 659. 664. Trunsport
in Biochemistry
(1978) 123. 285-286
Preliminary X-ray Study of Crystals of Human Transferrin
Transftarrin is responsible for iron transport to Iiclnoglo~)in-synthesizing red blood cells. Human transferrin is a glvcoprotein consisting of a single polypeptide chain of (j7G residues wit11 a molecular \\-eight of 81.000 (MacGillivray of al.. 1977). There MI’C t\vo itlcnt~ical cart)ohydrate chains with molecular weights of approximately 24(H) FL.II.+. I)oth of which are connected to asparagine on the peptidc hackbone 1)~ asparert al.. 1977). The protein is capai)k* gill~l-i\‘-;Lt~etvl-glu~osamine linkages (MacGillivrwy of binding two ferric ions tightly hut reversibly. Transferrin can also bind a varirt,! of othw metal ions. which sul)stitutta for ferric ions at thr two iron-binding sites (Putnam. 1975). Trwnsferrin is among the l)t~st-charactcrizeci of t hi mct.al t,ra.nsport proteins, in t,crrns of physical nnd hiochc~mical properties. biological function. iind int,eractions 1j.it,h tar@ wlls. Work towartl t,lrtA primar?; secptwt:t~ of human t~rwlsferrin is nearing conlJ)lt~tion (Sutton et rrl.. 1975). wd tlw cht~mical structurt~ of the t:arl)oh~dr>~tt~ chains has INYW rt~portt~tl (Spik rt ~2.. 1975). ‘Nit, rnimy ph~sicill and chtwical studies of t,r;wb(ibrrin II:\\-t, raiwd wvcraI fitIltlilttlr~nt,~~l Stl~ll~tUKll t{uwt,iolls t.llilt, might, best lNb illlS\Yl’t’l’tl l)y il cr?st;tllograpl~ic anal.ysi s of t,hr Jwot,t%l. ‘I‘htw 11;~slwtw pLt~~klJhJ intc~rt~st alld rontrovcrs~y concerning the iron-binding sitw irwluding questions al)out the t,ypt*s of amino acids that participate in t’htw sit,tbs: thta possihlr difftwntw lwtwt~t~11 the tn-o iron binding sites; the roles that anions. suc11 as hicarbonate, pIa> at thctsca sites: and the mechanisms t)y which various metal ions substitute for iron. Ttw crystal structure of transferrin should also Ilclp resolve t,he current, questions about possible internal structural homology wit’hin the molecule (MacGillivr;zy et t/l.. 1977). In addition, a high-resolution crystallographic: st’udy of t ransferrin may rtivwl thtl t~onformations of the carbohydrate moieties. which havtk bwn implicatt~d in ttw binding of transft:rrin to reticultqtc receptors (Aisw. 1!)74). (‘r~st,allograJ)hic studies of rill)t)it transftxrrin (Al-Hilal P/ (11.. 1976) irtltl of’ cllit:ktw tra~lsfrrrin (*J. E. ,1l~,I:t, Jwrsoni~.l comrnunic~~tion) arc in progress. H umall swum apotransfcrril1 11~s purch;~wd fronl Rrh ring\vtarkta C’ompany. ‘I’lrl~ nJ)ot~t~;ltlsft~l~l.irlevils corivt~rt~t~tl to transftlrrin 1)~ titrating solutions of ttltl protein in (I*-& RI-t)iwrtwnatt~ (pH 7.5) \\,ittl a11 iron complrx of‘ nit~rilotri;~t*t~t ic acid. ‘1’11thprottbin solut,ion was dia1yzt.d against a sodium Iblrbonate solut,ion t,o assurt’ rt~platwnrtrt of all rlit,t.ilot,r,i;lcetRtC anions 1)~ t)icnrt)onate. Thr solution wts t:ollct~ntr;~tt~tl t)y vacuutn dialysis against, 0#4 M-sodium nawd.ylat~r huffw (pH 5.75) t.o il prot.tbin conwntrat iwl of about 40 mg/mJ. Crpst)aJs WeJ’t’ obtained hy the batch method. using (I.3 to 0.5 nil portions of the protein solution, to which So (, polyet hylcne glvcot (average molecular wright 4000) was added. The solutions, stored at 4°C’. produced small crystals within 72 hours. Aft’er approximately ten days, red plate-like crystals with dimensions up to
286
L. J. DoLUC,zS,
F. L. SUDDATH,
R. :\. G.4318 .4ND C. I?.. BUGC,
1.5 mm x 0.5 mm x 0.4 mm were obtained. Precession photographs and diffractowith 11,: 78 a. h = 94 .f and meter data indicate that t,he cryst.als arc’ orthorhomhic. c =- 112 A. The space group is 1’2,2,2,. as indicat,ed hy bhe systematic absence of reflections hO0 with h odd, Ok0 with k odd and 001 with 2 odd. The diffraction data extend weakly to a resolution of 2.6 a on photographs taken with crystals in stationary orientations. The density of the transferrin cryst’als, as measured by the Ficoll gradient technique (Westbrook, 1976), is 1.193 g cmm3. Assuming that the unit cell parameters were unchanged in the Ficoll gradient. and that the protein has a partial specific volume of 0.725 cm3/g (Putnam. 1975). the calculated (Colman & Matthews. 1971) mass of protein per asymmetric unit is 84,884 daltons. which is not significantl! different from the molecular weight of 81.000 reported by MacGillivray (1977). Native diffractometer data to 6 p\ have been collecbed. We are currently searching for isomorphous heavy-atom derivatives. This research was supported by Unitsed States Public Healtll CA-13148, and DE-02670. Institute of Dental Research Comprehensive Cancer Centri Department of Biochemistry University of Alabama in Birmingham University Statioll, Birmingbarn Ala. 35294, U.S.A. Received 24 April
Services grants CA- 12 159,
L. J. D~LCCAS F. L. SCJDDATH K. A. (:Aiw c. E. Brrcc
1978
REFERENCES Aisen, P. (1974). B&t. J. Haematol. 26, 159--163. Al-Hilal, D., Baker, E., Carlisle, C. H., Gorinsky, B., Harsburgh, R. C., Lindley, I’. F.. Moss, D. S., Schneider, H. & Stimpson, R. (1976). J. Mol. Biol. 108, 255-257. Colman, P. M. & Matthews, B. W. (1971). J. Mol. Biol. 60, 163.-168. MacGillivray, R. T. A., Mendez, E. & Brew, K. (1977). Proteins vf Iron Metabolism (Brown, Aisen & Crichton, eds), pp. I33--142, Grunt & Stratton. New York, San Francisco, London. Putnam, E’. W. (1975). The Plasma /‘roteins (Pntnam, cd.), pp. 265 ~316. Academic Press. New York, London. Spik, U., Bayard, B., Fournet, B., Streckrr, (2.. Houquelet, S. & Montm~~il. .I. (1975). FEBS
Letters,
50, 296.-299.
Sutton, M. R., MacGillivray.
R. T. A. h Brclw, K. (1975). Proteins of lron Storage and and Il1edi&e (Crichton, ed.). pp. 19 ~26, Nort,h-Holland Publisbing Company, Amsterdam, Oxford. Westbrook, E. M. (1976). J. Mol. Viol. 103, 659. 664. Trunsport
in Biochemistry
