J. Mol. Biol. (1976)

101, 11-2-l

Structures of Triclinic Mono- and Di-N-Acetylglucosamine Lysozyme Complexes-A Crystallographic Study K.

KITRACHI~,

L. C. SIEKER of Biological

Department

ASD

:

L. H. JENSER

Structure

of Washington School of Medicine Scuttle, Wash. 98195, r’.S.A. University

(Received 10 June 1975, and in revised form

20 October

1.975)

N-acetylglucosamino and di-N-acetylglucosamine bind to lysozyme in the triclinic crystal form. At’tcmpts to bind t,ri-N-acetylglucosamine were unsuccessful. Difference synt,heses showed bot’h (:alNAc$ and di-GalNAc to be bound as the ,8-anomers, and revealed shifts in the positions of some of the lysozyme atoms. As was found in the binding studies with tet’ragonal lysozyme, the side chain of Trp62 moved toward the bound inhibitor. The carbonyl oxygen atom of Ala107 appeared to shift slightly, but t’herca was no suggestion of movement in the lobes of the molecules as was evident in the tetragonal crystals.

1. Introduction The structure of lysozyme in the tetragonal form has been solved by the method of multiple isomorphous replacement (Blake et al., 1962,1965,1967a; Corey et al., 1962; Stanford et al., 1962). Detailed descriptions of the conformation of the protein (Blake et al., 1967~; Phillips, 1966,1967) and its complexes with saccharides in tetragonal crystals (Blake et al., 19676: Johnson & Phillips, 1965) provide a basis for understanding the general nature and catalytic properties of the protein. Nevertheless, certain regions of the protein were not clear (Imoto et al., 1972), and it would be desirable to study the molecule in another crystal form. Moreover, saccharidebinding studies in another crystal form would provide confirmat80rp evidence for t,he mechanism of catalysis (Ford et al., 1974). In 1970 Joynson and colleagues (Joynson et al.. 1970) solved the structure of the t#riclinic form of lysozyme from the tetragonal by applying bhe rotation function co-ordinates are (Rossmann & Blow, 1962) at 6 A resolut3ion. Once approximate available for the triclinic form so that phases can be calculated. the molecular model can be refined in the usual crystallographic sense with data from the triclinic cryst#als. Refinement is in progress in this laboratory. and the conventional R (= zj& -- F,$zF,,) h as b een , reduced t)o 0.211 for the 1.5 A resolution data set. Using calculated phases we have determined the structures of the X-acetylglucosamine : lysozyme and di-1C’-acetylglucosamine : lpsozyme complexes in the lYlliversity of Washington, 7 Present address: Z)opartment of 13iochemistry, 98195, U.S.A. : .Abhrtwiations used: GalNAc. Iv-acetylglucosamin~~: LYZ, lysozymc. II

Seattle,

Wash.

K.

I”

KUHACHI,

L.

C. SIEKEH.

ASI)

I,.

H. JENSEN

t,riclinic crystal form. Since lysozyme is the first’ protein to have its stru&uro determined at high resolution in two different spact‘ groups, it’ is desirable to compare. details of the conformation of the bound substrat,e analogues as well a:: the conformation of the protein itself. I1~ defined by Blake et al. (19676), lysozyme has six subsites for substrate binding: A, B. C, D, E, and F, each corresponding to a saccharide unit, and running from one end of the active-site cleft to the other with catalytic groups Glu35 and Asp.52 between sites D and E. Binding at the A, B, and C sites have been directly observed by crystallographic methods involving binding of tri-GalNAct in t’etragonal lysozyn~ crystals (Blake et al., 19673) and D-site binding has been observed for the lactone moiety of a la&one derivative of tetra-GalNAc (Ford et aZ., 1974). Binding has not’ yet been directly observed in the E and F sites. although there is chemical evidence for F-site binding as shown by a dye replaced by hexa-GalNAc (Rossi et al., 1969). Although we were unable to obtain complexes of triclinic lysozyme with oligosaccharide bound at the D, E or F sites, the structures of GalNAc : LYZ and complexes reported here provide additional informat’ion for di-GalNAc : LYZ understanding lysozyme in its general and catalytic properties.

2. Experimental (a) Crystallizatio~l Hen egg-white lysozyme, lots LYSF 9BD and LY 9 FA (Worthington Biochemical Co.) was used without further purification for all experiments. To a solution of lysozyme in 0.05 M-acetate buffer solution (pH 4.5 to 4.6), solid NaN03 or a sample of 5% solution of NaN03 in 0.05 M-sodium acetate (pH 4.5 to 4.6) was slowly added with st’irring to a firm1 concentration of 1 o/0protein, 2% NaN03. The pH was readjusted to 4.55 with 1.0 M-acetic acid or NaOH solution. The solution was sterilized by passing it through a Millipore filter into clean glass dishes, but subsequently it was not,ed t,hat a solution without sterilization also gave good crystals. Bulky, rhomb-shaped crystals grow to sizes of 2 mm x 1 mm j( 1 mm in several months at room t’emperature. Crystals from LYSF 9BD were almost ideal in shape fol X-ray diffraction work, whereas those from lot LY 9FA were wedge-shaped. In either case the crystals were t)riclinic, space group Pl with unit-cell dimensions as listed in Table 1. The unit-cell volume is 26,200 -i3 and cont)ains one molecule. At higher conrryst,als tend t,o grow. centrations of NaNO, or at higher pH (4.8) monoclinic TABLE

1

Unit cell parameters b

cz (deg.) ~~_____.

(A) ~~__~~.

Lysozyme GalNAc : LYZ di-GalNAc : LYZ

27.283 (10) 27.244 (4) 27.266 (3)

31.980 (10) 31,909 (9) 31.898 (4)

34.291 (13) 34.349 (10) 32.244 (4)

81.53 (5) 88.42 (2) X8.50 (1)

B (deg. ) ~

108.57 (3) 108.53 (3) 108.38 (1)

Y (%.) ~~~.__

III.85 (3) 111.82 (3) 111.99 (1)

Values for native lysozyme are averages from 8 crystals and the estimated standard deviation? in parenthesis are calculated from the sample variance. Values for the complexes and their estimated standard deviations baaed on 12 reflections from one crystal. t See p. 11.

SACCHARIDE

BINDING (b) Cry&al

IN complex

TRICLINIC

LYSOZYME

13

preparation

Crystals of the complexes of lysozyme with saccharides were prepared either by soaking crystals of the native protein or by co-crystallization. Soaking of native crystals was done in solutions 0.1 M in GalNAc, 0.01 M di-GalNAc and 0.005 M tri-GalNAc at pH 4.55 (mixture of a- and fi-anomers). Crystals soaked in UalNAc and di-GalNAc solutions remained in good condition over an extended period, but those soaked in tri-GalNAc solution were fractured within 2 days. Precession photographs of GalNAc : LYZ and di-GalNAc : LYZ showed perceptible intensity changes. Co-crystallization of GalNAc and lysozyme was carried out, with GalNAc : LYZ in a LOO : 1 molar ratio. Protein and GalNAc were dissolvcld in 4 ml of 0.05 M-acetate buffor solution at pH 4.55 with Z”/b NaNO,. After sterilizing the solution by passing through a Millipore filter, the pH was adjusted to 4.55 with 0.1 N-acetic acid. In about 2 weeks, t,riclinic crystals grew t,o a size of 0.7 mm x 0.5 mm x 0.4 mm and remained stable for over a year. Precession photographs of cryst,als grown in t’his wap appeared itlent,icnl t,o those from crystals prepared by soaking. Co-cryst8allization of di-GalNAc and lysozyme was similar to t,hat, of GalNAc but with grown from rat#ios of di-GalNAc to prot’c,in of 1 : 1, 5 : 1, 10 : 1 and 20 : 1. Crystals solut.ions with ratios 5 : 1, 10 : 1, and 20 : 1 showed essentially identical intensity changes on precession photographs. Thcx crystal used for data collection was from the solution wit.h caoncclntrat,ion ratio 10 : 1, since cryst,als from the ra,tio 20 : 1 appeartd inferior in quality. An attempt t,o co-crystallizcl t,ri-GalNAc and lysozymc~ was from a solution similar to that. for GalNAc except with a saccharido to protein ratio of 1 : 1. Within 2 days, irrqular dodccahedral crystals of tetrnyonal lysozymt (presunlably the tri-GalNBc complex, s(b(’ Discussion) formed and in about 2 weeks triclinic crystals began to form. The t,ctragonal crystals grew to an approximate size of 0.4 mm x 0.4 mm. :< 0.5 mm in 3 weeks, remainitlg stable until they fractured and dissolved strcxral months later. The t,riclinic cry-stnls complex) rc~rnnined intact for ovrr a yc>ar. (preslunably t.hc GalNAc or di-C:nlh‘Ac (c) Data

collection

Intensity data were collrct,rd for the GalNAc : LYZ and tli-

Structures of triclinic mono- and di-N-acetylglucosamine: lysozyme complexes--a crystallographic study.

J. Mol. Biol. (1976) 101, 11-2-l Structures of Triclinic Mono- and Di-N-Acetylglucosamine Lysozyme Complexes-A Crystallographic Study K. KITRACHI~,...
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