Neutron diffraction study of carbonmonoxymyoglobin

J. Mol. Biol. (1991) 220, 381-399

Neutron

Diffraction

Study of Carbonmonoxymyoglobin Xiaodong

Chengf

Center for Structural Biology, Department of Biology Brookhaven National Laboratory, Upton, NY 11973, U.S.A. and Department of Physics State University of New York at Stony Brook Stony Brook, NY 11794. [/.&‘.A.

and Benno P. Schoenborn Center for Structural Biology, Brookhaven National Laboratory, {Received Neutron

Department Upton., NY

of Biology 11973, U.S.A

11 September 1990; accepted 2.2 February

1991)

diffraction

data from a crystal of carbonmonoxymyoglobin were refined by restrained least-squares procedure in reciprocal space, in conjunction with a solvent analysis technique, to a final R-factor of 11.3 o/O. The ligand CO occupies two sites and its binding conformations are distorted from the linear conformation. The N” atom of the distal histidine residue is deprotonated (not deuterated), and a water molecule is bound to the N’ atom of the distal histidine. The side-chain of Lys56 (D6) exists in two alternative charge-binding sites. His24 (B5) and His119 (GHl) share a hydrogen atom. His12 (AlO) and His36 (Cl) are deprotonated. The deprotonated imidazole ring of His12 (AlO) may act as a hydrogen-bond acceptor. The heme group is planar within 0.09 A rootmean-square (r.m.s.) deviation from planarity. The solvent environments for the two propionic acid groups are different. The side-chain of Arg45 (CD3) forms hydrogen bonds acid groups. An average with the side-chain of Asp60 (E3) and one of the two propionic N-2H . . 0 angle in helical regions is 147( + 11)“. Eleven main-chain amide hydrogen atoms from hydrophobic residues do not exchange with deuterium. The overall atomic occupancy factors for the main-chain and side-chain atoms are quite uniform, at @97( kO.07) and 0*93( *@lo), respectively, as shown by an occupancy analysis made at the end of the refinement procedure.

PROLSQ, a modern

Keywords: neutron

diffraction;

myoglobin;

1. Introduction

hydrogen

bonding

examined using a modern reciprocal-space leastsquares refinement (Hendrickson, 1985) together with a solvent analysis technique (Schoenborn, 1988; Cheng & Schoenborn, 1990). This paper focuses on five aspects of the structure of MBCO. The general refinement is described in section 2. The structure of the heme-CO complex and the local effects of CO bonding are analyzed in section 3. Section 4 gives details about the location of non-exchanging (or slowly exchanging) protons of the main chain and the protonation states of the imidazole ring of histidine residues. In the last two sections, 5 and 6, we discuss the hydrogen bonding and electrostatic stabilization based on the final refined co-ordinates of the protein.

carbonmonoxymyoglobin The structure of (MBCOS) was originally determined from neutron crystallographic data (Norvell et al., 1975) at a resolution of 1% A (1 A = 0.1 nm) and refined by a real-space method (Hanson & Diamond Schoenborn, 1981). This structure has been re-

t Present address: Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, Ir;Y 11724. U.S.A. 1 Abbreviat,ions used: MBCO, carbonmonoxymyoglobin; r.m.s., root-mean-square; XAKES, X-ray near-edge spectroscopy; OxyMB, oxymyoglobin; MetMB, metmyoglobin: n.m.r., nuclear magnetic resonance. 3X1

2. Reciprocal

Space Refinement

‘l’hft MKCO crystal

was grown from a sat,urattd ammonium sulfate solution at pH 57 in a pressure vessel containing CO gas and soaked in approximately 90 s<, 2H,0 mother liquor solution for several months to replace the wat,er (H,(j) of caqvstalization. Neutron diffraction data at a resolu1ion of 1.8 A were eoIlact.ed from a single carysial (Norell et nl.. 1975). The method of reciprocal space refinement is the stereochemically restrained-parameter least-squares procedure (PROLSQ) of Konnert (197(i), Konnert $ Hendrickson (1980) and Hendrickson (1985). The positional co-ordinates (zyz), one isotropic thermal exchangeable factor (for factor (H). one H/2H exchangeable hydrogen atoms only) and one occupanty factor (&) (for wat,er molecules only) were refined for every atom in the prot,ein and solvent, molecules. Only one variable occupancy, &, per water molecule is refined, while t.he atomic oc(~panty factors of protein atoms are kept ate & = 1. Each refinement cycle refines either just atomic C*Oordinated zyz. or refines zyz together with H, or f’ or 0 (for water molecules only). We simply alternate the application of zyz, f, II and & shifts in successive cycles. The least-squares refinement as described by (‘heng & Schoenborn (1990) improves the agreement between the solvent-modified observed struct,ure factors, F,,(h,k,l), and those calculated from the atomic scatt,ering lengths and the co-ordinates of the protein model, E’,,,(h,k,Z). The agreement between the observations (data) and the calcula.tions (model) ran be described in terms of t,he R-factor:

data. d where lPOml = IPobbs- FsO,l (for low-angle spacing > 3 8) and IF,,1 = I$‘& (for high-angle dat,a, of d spacing < 3 A). Fso, is the solvent component the protein crystal (Schoenborn, 1988; Cheng B Schoenborn, 1990). The numerical calculation of hi01 depends on the evaluation of the solvent, density m reciprocal space and does not depend on a Fourier flattening technique as used in PR(01,SQ and described by Wang (1985). Neutron Fourier maps (2/ibbs - PCal) were c:alc11lated based on the improved phases including the contribution from the shell solvent. and then the maps were examined on a graphics system, Evans & Sutherland PS300. using FRODO soft,ware (*Jones. 1982). These improved maps (Cheng & Schoenborn, 1990) allowed the easy placement of bound water and ions, and also suggested some changes in the surface structure of the protein. In all, 87 water (‘H20) sites and four ammonium ions (N2Hz) and one sulfate ion (SO:-) were located in t,hr course of t~he analysis. The localized water molecules were refined using restraints between water-protein and water-water associations. The refinement continued by alternating several cycles of least-squares refinement with an updated analysis of the shell solvent,, examination of neutron

Fouricxr, NliL})S. ~~lltl il ltl2Llfllitl rc~i)uiltlirl~ of sl(4k. chains antI solvent rIlr,l~YYllrs (ill I.hl. glx}‘hit~s system. After five su(~h itc~rat.ions t 111%i~c~lir~~~rrrt~nl stopped at a stage where the least~squarc~s rcifillc, men1 gave no furt,her drop in the K-fa(~tor. \vhic.lr had c*onvcrgcd to a value* of 12.7”,, \vlth ,~oo~ stereochemistry. Tight st,t?rcoc:‘rlc,irli(.ill rcbatr;li rrt s were used in the refinrmrrlt 1.0 rrdu(~(~ &siations of the modrl from ideal stt~reochemist ry. ‘I’hrb refirrcs ment was continued fhr (light rnor’cb c~yc~l~s 1%it h relaxed restraints, reducing the K-fac*t,or to t t ‘5”,,. A detailed analysis oft bra (‘0 confortna~t ion was t tlfhrl performed. The atomic occ*upanc+s of protein at<)nls were refined at the end of the refinr~mc~nt pro&urt~. keeping all other paramc~tt~rs af their rctinrbtl \.aItI(‘s. The final c.rq’stallograJ)hi(. K-factor is t 1.4 ()() MICI t tI(l r.rr1.s. error in bond lengths is 0.006 -9 li)r IIOII hydrogen bonds and 0~22 a for t~~drc)yc~ll~rt~Ia~c~tl bonds ((‘heng & S(~hoenborn. t 990) (a) Srrondar?y

strltc t 0 rf’

At an early stage of refinement. the six atoms of ii peptide unit ((‘7. (‘i, Oiz N,+,. Hi+ 1- (‘I,,) wert’ restrained to be coplanar. but after the K-factor dropped to 200/,, a five-at,om ((:I. (‘i. (Ii, Ni +1. (Is+ ,) coplanar peptide was used; this was redutzed further. after the R-factor fell below I 5(?o. t.o a four-atom (Cy, Ci, Oi% Ni+l) peptide plane with torsioil restraints to maintain a reasonable omega angle. The r.m.s. error in Jjlanarity of t)he peptide bonds ih 0.014 A. (b) Ilernv

rqfirwnwnt

The four pyrrole groups of the home were each separately restrained to be planar, but there was IIO restraint on the overall planarity of the heme group. The iron atom was not restrained to be in any plant-. though the iron-pyrrolr nitrogen distances wtarc restrained to MI A (Peng 8 Ibers. t 976). Two t,orsion-angle restrictions werr applied to two acsitl groups. The dist,anc:e of’ t hca propionic, heme-proximal hist)idine linkage from the N” iitOU1 of His93 (F8) t,o Fe \vas r&rainr~d at 2-10 x. ‘J’hgl only st~rrroc~hemic*al rcsstraints on t,hcL (‘0 ligantl involved the (‘-0 hontl length and I he tlistanc.ca between Fr and t,he ca,rborr at’om of the (‘f ) ligaiitl which where restrained to valurs of’ 120 .A and I.78 A, respcsctively. thtt Ftb (’ 0 bond irngSIc>was not rest.rained. No other st,ereoc~hrmicA restraint was placed on the l&and. densit) thr neutron ShO\VS Figure t binding (FobsFC’cal> a,,,) at 0.30 .1+./A” at thtb tigand site derived from t,he structure factors ( P,.a,, s(,,,) calculated from the co-ordinates of the final retincatl protein struc:t,urr otnittirlg the (‘0 lipancls. Ttlt* shape of the Fourier peak at t.he ligand site suggests more than one (10 ligand conformat,ion. Multiple (‘0 tigand conformations were also observed by infrared spectroscopy (Makinen et (II., 1979; Brown rt n/.. 1983). One goal of our carystallographic< refinement was to delin&te possible mutt,iple &es of t.he ligand.

Neutron Diffraction

383

Study of Garbonmonoxymyoglobin

(b)

Figure 1. Srutron density for CO ligand. The calculated structure factors do not, include (‘0. (a) Side view: the d&al left,) and proximal (lower right) histidine residues are shown. (b) Orthogonal view; pyrrolr rings of the hrme A. K. (’ and 1) are do~vn. right, up and left,. respectively. (upper

PROLSQ occupied

was therefore modified to allow partially ligands. Two atoms of a particular (‘0 molrcule had thtx sa.me variable occupancy. &? and t hr oc+cAupancGesof the two observed conformations were constrained to unit,y: i.e. Q2 = I- /&. To avoid coupling between occupancy and temperature factors. shifts for the two factors were a,pplied in alternate cyc~lrs.

3. Heme Geometry and Environment Tuo (‘0 ligantl orientations were furt,hrr refined leading to occupancies of S&2’& and -tl%(!,,. The refined t.wo conformat.ions of t.he (10 ligand arc shown in Table 1 and Figure 2. with an Fe-(’ distance of 2.13 a; the Fe-GO angles of t.he two conformations are 143” and 130”: thr average

Table 1 Geometry of heme with CO ligand Atom pair

Distance (A)

Fe

(’ (Ilganti)

213

E’cx Fe to Fe to Fe.

0 (ligand) hem plane nitrogen plane SC2 (FX)

3 19. 348 0~10 0~06 226

I)istanw 0B ( ’ (ligand) () (ligand) N”’ (F8) N” (F8) N”’ (F8) SE* (E7) SC2 (E7) I)irwtirm

hemr normal

0 (main F1) 0’ (Ser F7) (I (ligand) Cl (igand)

wsinrs

Hemr plane Sitrogrn plnnr

(b.58 1.33, 0.99 (I.19 2x5 3.16

Anglr (0) Fe

(‘-0

Tilt angle of proximal imidazole ring from a plane through N”-Fe-S’ Dihedral angle between heme plane and proximal imidazole ring Dihedral angle between nitrogen plane and proximal imidazole ring Angle with hrme normal

Nd’-‘HJ1 Nd’-‘H6’

0 (main F4) Oy (Ser FT)

3.1 1

348. 2.60

of plane normals (related to orthogonal I - 0.895 - 0909

co-ordinate):

O&I 0.385

?l -- ().“7:! -1 ~ 0~158

X. (.‘hang

and

B. I-‘. Bchornhorn --

(b)

Figure 2. Different conformations for the CO ligand. (a) Stereo view (side) of the heme group and ligand CO. with the distal histidine residue on the right side and the proximal histidine on the left side. (b) Stereo view (orthogonal) of the heme group and ligand CO. The 3 nearest neighbor residues to the CO are Phe43 (CDl) (lower right), His64 (E7) (upper right) and Val68 (El 1) (upper left). The proximal histidine residue (not shown) is down to the other side of the heme. The various distances and angles are listed in Table 1.

B-values

are

The R-factor (II)

value

16.10

8’

decreased of

and

16.38

A’,

respectively.

by 0.1 y. while

CO decreased

by 2 8’

from

the average the

single

CO model. The R-value of Fe is 15.23 8’. While these changes in R-factor and R-value are small, they suggest that two alternative CO ligand positions are more likely than one. The geometry of the Fe-C-O bonds in this strut ture differs significantly from those in free porphyrin model componds; for example, the strut ture of pyridine Fe(II)CO tetraphenylporphyrin (Peng & Ibers, 1976): with a linear Fe-C-O arrangement, and an Fe-C distance of 1.77( +@02) A. Generally, the observed distorted ligand position is caused by the steric hindrance from the two hydrophobic groups, Phe43 (01) and Val68 (El]), and distal His64 (E7) (Fig. 2(b)). The carbon atom is displaced off the heme normal by 0.58 A, in the direction between pyrrole rings A and 1); the angle with the heme normal is 16”. The oxygen atom is displaced by 1.33 A and 0.99 A, respectively, from the heme normal. The two conformations of thr CO ligand may correlate to alternative configurations observed in other parts of the protein that exhibit a similar distribution of occupancy factors; for example, the two conformations of the side-chain of

Lys56 (D6) (see Fig. 5), and the hydrogen atom shared by His119 (GHl) and His24 (B5) (see Fig. 6). Table 2 compares this CO structure with other CO ligand analyses. The erythrocruorin structure has an Fe-C distance of 2.4 A, an Fe-C-O angle of 161( + 5)” with the iron atom within 0.01 A of the heme plane (Steigemann & Weber, 1979), while resonance Raman spectroscopy reveals an Fe-C

Table 2 CO bond distance and angle changes CO-heme structures

Year

Method

Reference

1S90 1986 1985 1984 1984 1981 1979 1976

Neutron X-ray XANES EXAFS Kaman Neutron X-ray X-ray

This work Kuripan r/ al. Bianconi ct al. Powers et al. Yu et al. Hanson et al. Steigemann rt al Peng & Ihers

t Two conformations

in different

Fe--(’ distance (4 2.13 227

Fe x-0 angle (“1

143, 1307 141. 12ot 150 1.93 * 0.02 127+4 1.8 169+5 I.78 153 24 161+.5 1.77 f o+% 17S_+%

of CO are modeled.

Neutron D#raction

Study of Carbonmonoxymyoglobin

bond of 18 A and a Fe-C-O angle of 169( f 5)” (Yu et al., 1984). EXAFS measurement gives a value of 127( +4)” for the Fe-C-O angle, and observed a correlation between an increase in the Fe-C bond length and a decrease in the Fe-C-O angle (Powers near-edge spectroscopy et al., 1984). X-ray (XANES) suggests that the Fe-C-O angle is 150” in MBCO crystals and in solution (Bianconi et al., 1985). The X-ray structure of MBCO presented by Kuriyan et al. (1986) shows two CO ligand conformations, with Fe-C-O angles of 141” and 120”, and an Fe-C bond length of 2.27 A. These data differ significantly from observations of carbonmonoxyhaemyoglobins A and Cowtown as reported by Derewenda et at. (1990), in which the Fe-C-O group is bent from linearity only by about 7”. (a) Heme and nitrogen planes With a least-squares plane of the 24 central porphyrin atoms defined as the heme plane, the iron atom is within 910 A of the plane in the CO-myoglobin complex, 0.40 A from the plane in the X-ray structure of metmyoglobin (Takano, 1977a), and 916 A in the neutron structure of metmyglobin (Cheng, 1989). The nitrogen atoms of pyrrole rings A and B, NA and NB, are displaced out of the plane towards the proximal side by 616 A, and the nitrogen atoms of rings C and D, NC and ND, are set towards the distal side by 096 A from the heme plane. The propionic acid groups of pyrrole ring C tip towards the proximal side and those of pyrrole ring B tip towards the distal side. The two vinyl groups of pyrrole rings A and D are twisted out of the plane with the CB atoms 64 A and 0.8 A from the heme plane. The iron atom lies within 606 A of the least-squares plane of the four porphyrin nitrogen atoms in the CO-myoglobin complex, but 927 A from this plane in the X-ray structure of metmyoglobin (Takano, 1977a), and 609 A in the neutron structure of metmyoglobin (Cheng, 1989). All four pyrrole ring nitrogen atoms are exactly in the nitrogen plane. The dihedral angles between the heme plane, the nitrogen plane and the imidazole ring of the proximal histidine residue are 91” and 94”, respectively.

385

The early real-space refinement of the COmyoglobin complex showed that there was no hydrogen bond between CO and the distal histidine residue, and that NC2 atom is not deuterated (Hanson & Schoenborn, 1981). This important feature of the distal histidine residue was carefully re-analyzed in our refinement and was confirmed. The refinement started with zero scattering (b = 0.0 when f = 6355) of the ‘HE2 atom. With an increased occupancy, the 2Hc2 was pushed away from its stereochemical position while the CO ligand was pushed in the opposite direction. In this case, the ‘HE2 atom reached a scattering length of only 042 Fermi units, as compared to the expected value for full occupancy of 667 Fermi units. Refinement was continued after removing the 2HE2 atom from the distal histidine residue; the final distance from the NC2 atom to the superimposed carbon atom of the CO ligand is 3.11 A, and to the oxygen atom 3.48 A and 2.60 A, respectively. The deprotonation of the distal histidine residue at pH 57 agrees with a pK value of 590 as determined by Friend & Gurd (1979). Neutron diffraction studies have shown that the distal histidine residue is hydrogen bonded to the ligand in both met- and oxymyoglobin, but not in MBCO (Norvell et al., 1975; Phillips 8: Schoenborn, 1981; Hanson & Schoenborn, 1981). A water molecule sits nicely between the 2Hd’ atom of the distal histidine residue, the Oy’ atom of Thr67 (ElO) and the side-chain of Arg45 (CD3). This water site is at a location described as a sulfate ion in early X-ray studies on metmyoglobin and deoxymyoglobin by Takano (1977a,b). The observed neutron density of -956 F/A3 at this site corresponds to a 2H20 molecule, since it is much higher than the scattering density (926 F/A3) of a sulfate ion. The sulfate ion is also absent from the X-ray structure of MBCO (Kuriyan et al., 1986); but a water molecule is confirmed in the neutron structure of metmyoglobin (Cheng, 1989). The location of this water molecule linking Arg45, His64 and two observed ammonium ions is probably connected to the flexibility of the distal histidine residue as suggested by Johnson et al. (1989).

(c) Propionic acid groups (b) Proximal

and distal hi&dine

residues

The Fe-N”* (proximal histidine residue) distance is 2.26 A. The NE2 atom is displaced 619 A from the heme normal in the direction of pyrrole ring B, and the angle with the heme normal is 5”. The tilt angle of the imidazole ring from a plane through NA-Fe--NC is within 4”, which is significantly different from the 19” found in the X-ray analysis on metmyoglobin (Takano, 1977a). The 2Hd1 atom of the proximal histidine residue shares a hydrogen bond with the carbonyl group of peptide F4 and with the hydroxyl group of Ser92 (F7), and these two hydrogen bonds help to hold the histidine ring rigidly in place (Fig. 3).

The propionic acid group attached to pyrrole ring C folds back into the proximal histidine side of the heme to form two hydrogen bonds with Ser92 (F7) and His97 (FG3). One ammonium ion and three water molecules are directly bound to the propionic acid group of pyrrole ring B, which folds up towards Arg45 (CD3) to form one water-bridge to its peptide carbonyl group and a hydrogen bond with its sidechain. Three water-bridges are formed between this pyrrole group and the main-chain atoms of Lys42 (C7), Asp44 (CD2), and the imidazole ring of His97 (FG3). The solvent environments for these two propionic acid groups are completely different: no water is bound to the proximal side. but. wat,thr molecules

and ions extend across the exposed edgt~ of the herrrt~ on its distal side, as well as to t.he surface. close, to the entrance to the heme pocket.

((1) Ilr~mw ~nzironmunt In all, 19 residues and two symmrt,ry-related residues are in contact’ with the heme atoms with distances shorter than 34 L$ (Table R). Of the 21 and Ifi are residues, five are basic hydrophilic hydrophobic or neutral residues. Of t,he total of 56 atoms in contact with heme, 43 are hydrogen, six non-H/‘H atoms. and only seven deuterium, Clearly, hydrophobic interact)ions and hydrogen atoms play a significant. role in stabilizing the heme. The (; helix contributes the largest. number of contacts. followed by the E helix, F(G corner and F helix.

4. Thermal Parameters, Hydrogen Exchange and Disordered Side-chains (/t) ‘l’he il\-er;tg,rt* is()tr()f)i(’ t.h
Neutron

Dijfraction

387

Study of Carhonmonoxymyoglobin

Table 3 Contacts shorter than 3 A between protein and heme atoms Number

Residue Sumbrr

Type

Itepion

Globin

atoms

Hrmr

atoms?

3.36

(b)

1

A .--_

0

chain atoms of hydrophobic residues are general]) onlv slightlv larger (1 to 2 A2) than those of the marn-chain ’ atoms. Surface hydrophilic residues often show la~gc~r mean temperature factors (up to 5 A’) than that for the corresponding main-chain atoms. The average (IZ) values f&r helices A through H arc’ 19(+5) A2, 16(&4) a’, l5(f6) ‘h2. Il(f5) .A’, 15(+4) A2, 18($-6) .h2, 17(+5) A2 and 17( +4) :i2, respectively. The average (K) values of t’he 25 central porphyrin at,oms of the heme (including Fe). and the imidazole rings of the proxirnal and distal histidine residues are 15.2( kO.3) A2, 14.2( 40.1) ,A2 and 16.2( kO.3) A2, respectively. (a) Kxrhangrcrhlr

hydrogen

and deuterium

atoms

The scat.trrinp length of an exchangeable hydrogen atom is given by the relation: b = (6.67 + 3.74)f -3.74. where 6.67 and -3.74 are the scattering lengths. in Fermi units, of deuterium and hydrogen rrspect.ively. and f’ is the fraction of deuterium present. The permissible range off is from 0 (hydrogen) to I (deut.erium). The scattering length is zero at .f= 0.36. and this is a match point with an uncertainty whether a hydrogen atom with 36% exchange is present or whether this site is vacant. The observed standard deviation for H/‘H exchange rates is as high as @25. There are three classes of main-chain amide H/‘H atoms: completely unexchanged cf I (blO), partially exchanged (0.11 0.62). The crystal was soaked in 2H20 solution for several months before data c*ollection. In all, 1I (7’5;) of 149

BCDE __ 40

,-.--

FG -_

~-

80 Residue number

.~ 120

(0) 160

Figure 4. Variation of the isotropic thermal factor (B). the hydrogen exchange f, and the occupancy (Q) as a function of residue number. The helices are indicated at the bottom of the Figure. (a) The isotropic thermal factor. The filled circles indicate the average temperature factors of main-chain atoms. and the open circles that of sidechain atoms. The last 3 filled circles refer to the heme and the :! CO conformations. (b) The fractional exchange of main-chain amide hydrogen atoms. The scattering length is zero at the value of @36. (c) The a.verage orcupancy of side-c-hains hevond the C” atom.

amide H atoms are completely unexchanged, 85 (57 ?;, ) are fully exchanged! and 53 (36%) are part,ially exchanged. (We note that the observed actual 2H20 concentration is 800/b; Cheng & Schoenborn. 1990.) Columns 4 and 5 of Table 4 give the observed exchange ratio of the main-chain amide group and the class to which it belongs. The distribution of the exchange ratio of the main-chain amide hydrogen atoms as a function of residue number is shown in Figure 4(b). The ratios show that the short helices C, D and F exchange more rapidly than the long helices A, H, E, G and H. The corners between helices and terminal residues seem to exchange rapidly, agreeing with the observation that these caorner regions have larger thermal factors and are more mobile. Table 5 lists the 11 completely unexchanged main-chain amide hydrogen atoms with deuterium occupancies less t,han 0.10 and the helical region they belong to. All of these 11 residues

388

Main-chain Residue number and type

H/‘H exchange

N

(’

I 2 3 4 5 6

Vat Leu Ser Glu NJ GlU

NAl NA2 Al A2 A3 A4

0.67 (>46 0.67 0.82 0.50 085

F P F F P F

ZH ZH ZH 2H 2H ZH

0 0 0 0 0 0

7 8 9 IO II 12 I3 I4 I5 I6

TOP Gin IRU Val t/Au His Val Trp Ata Lys

A5 A6 A7 AX A9 A10 All Al2 A 13 AI4

0.57 0.99 072 woo 0%~ 092 0.72 090 0.31 0.74

P F F 11 F P F U P F

2H ZH 2H H 2H ZH 2H H 2H *H

0 0 0 0 0 0 0 0 0 0

17 IX

Val (ihl

Al5 Al6

0. t 2 031

P F

H 2H

0 0

I9 PO 21 22 23 24

Ala Asp Val Ala GIJ His

AU1 ut u2 B3 u4 135

0.61 0.65 0.85 @70 036 W89

P F F F P F

2H 2H 2H 2H ZH 2H

0 0 0 0 0 0

25 26 “7 28 29 30 31 32 33 34 35 36

Gly Gin Asp lte Leu Ill? AX Leu Phe Lys ser His

136 87 I38 us BlO Bll El2 Bl3 1314 I315 IS16 Cl

0.7 1 062 0.85 0.22 0.07 090 0.37 009 013 043 0.61 0.73

F F F P u IT P 11 P P P F

2H 2H 2H 2H H H ZH H H 2H 2H 2H

0 0 0 0 0 0 0 0 0 0 0 0

3.21/%34/148 3.24/2.33/148 3.13/2.34/137 3.27/2.46/140 3.15/229/146 2.99/2.17/138 3.16/2.22/166 3.13/2.33/136

37 38 39 40 41

PiW (:lu Tbr La (Zlu

(‘2 (‘3 (‘4 c5 (‘6

w75 0.40 0.48 0.62

F P P F

2H 2H 2H 2H

0 0 0 0 0

3.31/2.53/134 3.13/2.36/132 3.1 l/229/143 3.22j2.271161

42 43 44 45

Lys Phe Asp Arg

(!7 CD1 (!D2 CD3

0.99 @42 0.39 0.81

F P P F

2H 2H 2H 2H

0 0 0 0

3.37/2.43/156 3.41/2.48/157

46 47 48 49 50

Phe LYS His Leu LYS

CD4 CD5 CD6 CD7 CD8

0.94 0.67 096 0.65 0.51

F F F F P

2H 2H 2H 2H 2H

0 0 0 0 0

317/2.36/140

51 52 53 54

Thr Glu Ala Qlu

Dl D2 D3 D4

0.40 0.85 0.77 0.29

P F F P

2H 2H 2H 2H

0 0 0 0

32712.351152 3.15/2.26/150 3.30/2.44/147

55 56 57 5x

Met LYS Ata Ser

D5 D6 D7 El

0.98 Ia0 @98 O-83

F F F F

2H 2H 2H 2H

0 0 0 0

3.41/254/147

325/2,40/ 146 3.08/2.21/154 3.49/2.58/k% 3.37/2.54 I40 3,13/2.26/150 3,14/232/141 3.43/2.54/ 150 3.00/2-28/ 130 2.91/2.25/124 3.48/2-56/154 3.36/2.53/140 3.16/2.28/153

O?

‘H-SC, (Lys79)/3.10/2.41/121

0” ‘H-X(3, main)/3.40/2.44/163 0” 2H-N’,(Lys133)/2.98/2.07/154 NE’-‘HE’ O(t. main)/3.45/2.69/131

NC2

Od’(Aspl22)/352

N”--‘H&l

Ob~(C:lu18)/~20/2~70/l 12

NC,-‘H Nc+mm’H

334/232/174 3.02/222/140 3.29/2.46/142 3.07/2.17/151 3.17/2.31/147

3.23/2.37/144

O~‘(Aspl22)~~17/1~40/12X Od2(Asp122)/2~91/201/148

0” OE2

(Trpl4), O?’ N<(Lys77)/2.69 Nc,( # Lys50)/:+65

061

Nr:2(Argll8)/3.04

N6’-‘Hd’ O(20, main)/2%S/l.93/158 N” 2H’2-N”2(Hisl19)/2~85/l~93/153 NE2-2HL22 0(56, main)/3.34/277/117 Od’ N’,(Lys56)/344

N”’

OY(Ser35)/3.15. NV2

OY(Ser35)/I+25

N\ O?(Glu52)/558 Oy (Arg31) Na’-‘Hd’ W~(Glu38)/413/3~40 Nd’-ZHal OE’(Glu38)/4.38/3.66 02 2H-X~+(#Lys79)/2~31/l~57/12:~ OY’~~‘Hy’ O(36, main)/2~71/1~77/175 oy N\( # Lys77)/313. 0: N$(Lys47)/521 NC+ ‘H O(98, main)/2.79/192/140 0’ Nz, (Lys47)/492 NE,m2H’ Od’(Asp60)/2~99/1.98/161 N;2-2H”21 @!(Asp60)/325/2.30/167 Nt#LZH”‘l O?( 1.54, heme)/333/251/14 + N’, (Asp44), X;, (Glu41) N$‘H’* O?( #Glu52)/414/3.48

3.33/2.46/ 146

N\~-‘H 0(47, main)/364/2.84/134 NC, (#Glu18), N’; (Glu54)/7.01 Oyl ‘H-N(54, main)/339/2.43/159 Of (Lys34), OE’ Nr, (Lys56)/4-74 O? 09

‘H-N(50, ‘H-N(51,

N$

(Asp27), N$

W

‘H-N(61,

main)/2.97/2.05/151 main)/3.32/2.41/157 (Glu52)

main)/337/2.48/151

t

Neutron Difraction

389

Study of Carbonmonoxymyoglobin

Table 4 (continued) Main-chain Residue num her and type

HfH exchange

s

(

Side-chain

.59

Glu

EL!

o+io

F

ZH

0

2.9.512.18/137

60

Asp

61 62

I&U

E3 E4 Ri E6

0.65 082

IH ZH ‘H ZH ZH LH H ZH ZH 2H 2H 2H ZH 1H H 2H 2H ZH

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3041’2,18/143 2.99/2.09/ 146

0.66 090 0.37 0.03 0.95 0.65 0.18 0.42 090 0.39 lb27 072 013 0.57 0.47 0.45

F F F F F 1’ u F F 1’ I’ F I’ I’ F I’ I’ I’ I’

083 0.99 067 0.8 I 1.oo o-69 0.98 086 0+30

F F F F F F F F F

ZH 2H 2H 2H 2H ZH lH 2H ZH

0 0 0 0 0 0 0 0 0

F F F F F

ZH ZH LH lH ZH

0 0 0 0 0 0

P P F

ZH zH ZH

0 0 0

63 64 6.5 6ti 67

1,) s l.ys His (:I> Val

E7 EX E9

El0 El1 El2 E 13

1.oo

75

ThI Val Leu Thr Ala l&U ( iI?, Ah IIC

i6

lxu

77 7x

Lys l+s

79

EF” EF3 EF4 EF5 EF6

87

Lys Glp HN HIS Glu Ala (:lU lxu I+

8X 89 90 91 92 93

PI-0 LtTl Ala GIlI SW His

F3 F4 F ‘j FS Y7 FS

098 0.98 0.90 095 075

94 95 96

Ala Th r I+

F9

06 1

F(;l I4:2

1.OO

tiH 69 70

71 72 73

i4

x0 XI X2 83 x4 x5 X6

97 9x 99 loo 101 102

HIS LYS I Ita Pro llt I+

103

Tyr l&U Glu Phe llr Ser Glu Ala IIts lita His Val Lrll His SW A% His

104

105 106 107

108 109 110 111 II2 I13 114 115 116 117 118 119

El4

El5 El6 lcli El8 El9 E20 KVl

EF7 EFX

Fl F2

G4 G.5 G6 t:7 (23 (i9

UIO 01 I (:I:! (iI3 G14

Gl5 (iI6 Gl7 (:I8 Gl9 (:Hl

0.78

*H

F F F

*H ZH 2H

0.29 0.72

P F

,?H ZH

0 0 0 0 0 0

O%O 004 @87 (k72 037 0.41 O-60 062 0.00 0.12 0.65 0.26 0.99 W60 0.73 0% 0.79

F U F F P P P F IT P F P

?I H ZH 2H ZH lH 2H ZH H H ZH 2H H 2H 2H 2H ZH

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

IJ P F F F

3,25/2.42/139 3.24/2.38/145

415/2.15/169 324/2.27/154

Of Nc,( Lys62)/4.60, 02 N’H$(209)/2.89 Od’. Odz (Arg45). 06’ Ni, N\-2H NC2

Ns,( Lys63)/440

(Glu59) 0(# 19, main)j2.51/l..i3/161 C/3,11. X”* O/3,48. 260

307/2.19/147 3.13/2.24/144 3.07/2.25/139 300/225/132

329/250/136 3.43/2.63/137 3.17/2.18/173

0” -*H”

O(66. main)~2-6i/l+Cj/l40

323!2,45/134 :~05'2~17'148 " /

N’, NC, N’, N\

309/%21/152 3.19/2.28/156

N;2-‘H”* 062(Aspl4l)/~lU/2~24/153 O? N’H:(N-Val)/433 0”.

282/1.96/143 3.10/2.19/154 3.12/2.28/141 3.21/2.37/148 3.12/2.43/ I30 3.28/2.34/161 3.03/2.12/161 2.89/190/173

.(Glul8), Ni, (#Glu41) W:(Glu85)/3~21 O?(Glu85)/3~28 (Glu4), N\ (#Glu38)

O?

N:+ N\

(Lys78) CY~(Glu148)/506, COO-(C-Gly)/5.94

(IL’ 2HdZ2-N62( #Asnl32)/3~50/2~91/117 OY-2HY Ocz2(154, heme)/2.75/1.80/175 N”’ Fe/%26 X6’--‘H61 (~y(Ser92)/~16/~51/123 N61 -*Ha’ O(89. main.)/2~85/2~20/121

-vi,

.tt~(#Glulo9)/2~59.

Nr, oy #GlU109)/3~17 N~-‘HP2 O!**(154, heme)/3.31/2.40/145 NT+ NS,(Lys42)/5.99 312/228/145 3,33/2+X/135 322/2.45/134

315/2.32/142

317/234/141 410/2.30/139 312/2.26/149 2.95/1.95/170 32612341157

3.10/2.14/158

Xi, . ..O"(Glu105)/2~54. Ni, . ..0't(G1u105)/340 X'i, Of(#Glu148)/3$2

O’-‘H”

0( # 77, main)

O?‘, 02

(LyslO2)

Oy~2HY O?‘. 02

O(104, main)/272/1.85/149 (#Lys96), (# Lys147)

3.12/2.20/157 300/2~08/154 310/2.44/125 3.04/2.11/156

309/212/168

309/2.31/135

NT .Ne2

(Asp20) (His24)

Table 4 (continued) Residue number and type

H/‘H exchange

1’4 125 126 I27 128

Phr GIJ Ala Asp Ala Gin

GH2 GH3 GH4 GH.5 GH6 HI HZ H3 H4

0.32 074 025 0.53 0.66 082 0.38 0.94

I’ F 1’ t’ F P P F

ZH 2H 2H 2H ZH 2H 2H LH

0 0 0 0 0 0 0 0 0

129 I30 I3I I *7”I33 134 I35 136

Gly Ala Met Asn Ly” Ala I&u Glu

H5 H6 H7 H8 H9 HlO Hll HIP

0.28 080 0.53 0.67 W61 0.07 0.18 0.49

P F I’ F P 1 1’ P

*H 2H ZH 2H ZH H 2H 2H

137 13X I 39

Leu Phe A%

H 13 H14 HI5

000 0.31 023

I7 P P

I-10

1.w

HI6

049

I41 142 143 144 I45

Asp Ile Ala Ala Lys

H17 H18 H19 H20 HZ1

I46

Tyr

I47

I PO I”1 122 I 23

Pro (:I$ ASP

0”

(Hisl2).

3.16/2.27/154 2.94/2.14/136 3,41/2.48/158 2.99/2.16/139 3.08/2.17/150

(f2

N’H;(178)~234

0 0 0 0 0 0 0 0

328/2.36/161 330/2-38/ 143 3.24/2.37/149 3.08/2.19/149 3,17/2.38/135 3,25/2.4.5/ 138 2.98/2.16/139 304/2.24/138

X62--2HdZ2 O~2(Olu136)/~~~12/2~5:~~lI6 NC+ (Glu6)

H 2H 2H

0 0 0

:924/232/ 159 3.09/2~28/ 137 3~l9/2~28/149

P

2H

0

3.06/2.27/137

092 0.31 0.14 0.63 0.63

F P P F F

2H 2H H *H 2H

0 0 0 0 0

3,47/2.55/152 2.88/2.05/140 3.08/2.12/165 3.44/2.55/154 3.43/2.59/l 40

H22

0.37

I’

2H

0

Lys

H23

090

F

2H

0

3.00/2.34/ 125 298/2,43/ 117 3 12/2,22/ 149 :+16/2,33/144

14x

Glu

I49 I50 151 I52 I 53 I54

I&U

H24 H(:l HC2 HC3 HC4 H(:5

0x4 0.73 0.54 @75 048 0.93

F F P F P F

2H ZH ZH ZH 2H zH

0 0 0 0 0 0

My Tyr Gin Gly Heme

(I”,

OdL

(L,vsl6)

NrZ-2HL2* Oczl( # I54 heme)/3.57/2.86/ I28 ’

0” 0’:

(Asn132) =H”= -N~+2(Argl:~9)/4~61/4~O0

N”2 .zf,J@ 0( # 147. main)/%4X/l-Xi/145 N’- 2Ht ;;(# 147, main)/3+2/2.24/128 N; - 2H O”(‘ryrlfil)/:j.46/2.4~/169 Nz, .061(Aspl41)/455 0”’ (His%). Oh2 N2H~(21X)/2~91

NC+ w-(174)/3.55. NC+ O~(Glul48)/440 W2H”. O(99. main)/%~7l/I+i6jl43

N’; NC,

iP 0” (XM)‘. O832 OCZ2 ()C21

0”!(#~:lu109)/3~40. O?( #Glu109)/4+3 (Lys87). o’* (#Lyslo?.?).

(I,,ssl45)

(Lysl40)

(Arg45) (SerSS), His97) (#Gln128)

(‘olumns 4 and 5 give observed hydrogen exchange, and classification into 3 classes: F, full exchange (20.62); P, partial exchange (01 I to (k61); U, no exchange (<@lo). The main-chain hydrogen bonds (N-‘H 0) are listed in column 8. The number hefore the Ist, solidus is the non-hydrogen distance (N 0); the number after the 1st solidus is the distance between deuterium and oxygen atoms; and the number after the 2nd solidus is the angle at the deuterium atom. The side-chain related hydrogen bond lengths (5 3.5 A) and angles or charge-charge distance are given in the tast column. The symbol # represents a neighboring symmetry-related molewlr.

are hydrophobic: two Ile, three Val, five Leu and one Trp. All unexchanged amide groups lie on the inside faces of helices A, B, E, G and H, facing into the hydrophobic interior of the protein. The peptides that have unexchanged amide hydrogen atoms have local minimum B values: for instance, Trp14 (A12) has the lowest B value within the A helix, and Leu29 (BlO) has the lowest B value within the B helix. Table 5 lists the average exchange rates by residue type, low exchange occurring only in hydrophobic residues. The average exchange for Ile (27%) is quite low relative to that for Leu (45%); possible explanation was discussed by Phillips (1984). Ile has a bulky side-chain with a

B-branch, allowing only one value for a helical x1 torsion angle. This confers extra rigidity on the helix at this point, and imposes steric hindrance on the neighboring peptide. Polar residues (Tyr, Thr, Asn and Ser) have a slighly lower H/‘H exchange than charged residues (Asp, Lys, Glu and His). Table 5 compares the difference in hydrogenexchange behavior with the neutron data from different myoglobin derivatives, i.e. CO-myoglobin, oxymyoglobin (Phillips, 1984) and two metmyoglobins (Schoenborn, 197 I ; Raghavan & Schoenborn. 1984). We emphasize that the four data sets differ in conditions of crystallization, refinement procedures used, the level of refinement and, particularly, in

Neutron Diflraction

391

Study of Carbonmonoxymyoglobin

Table 5 Unexchanged amide H atoms Avemge main-chain amide hydrogen exchange in MBCO

Main-chain residues showing low hydrogen exckznge in MBCO, MB02 and MetMB MBCO This study Residue

MBO, Phillips (1980) Unexchanged

MetMB Raghavan & Schoenborn (1984) amide H atoms

MetMB Schoenborn (1971) Residue

Number

Type

Region

11t

12$

2§

10 II 14 17 28 29 30 32 33 66 69 72 75 89 104 107 111 I12 115 134 135 137 138 14% 143

Val

AS A9 Al2 Al5 B9 BlO Bll B13 B14 E9 El2 El5 El8 F4 GS G8 G12 G13 G16 HlO Hll H13 H14 H18 Hl9

o4lo 030 PO0 0.12 0.22 @07 0.00 009 013 0.03 @18 0.39 @I3 0.98 0.04 @37 PO0 0.12 0.09 0.07 0.18 @OO 0.31 @31 014

@30 0.92 @43 919 0.09 0.00 WOO 000 921 0.42 0.00 042 @02 @80 I.00 000 055 0.17 047 035 0.00 025 0.00 0.00 0.00

0.37 034

7 36 57

8 21 71

LeU

Trp Val Ile L4Z.U

Ile Lell

Phe Val Leu Leu He Leu I..eu Ile Be Ile I.eu Ala Leu I..eu Phe Ile Ala

No exchange (%) Partial change (%) Full exchange (%)

1311

0.63 0.42

J

Number observed

Mean

Ile Trp Val Leu Phe Gly Ala Met

9 2 8 1x 6 11 I7 2

0.27 0.29 0.41 045 @46 053 0.62 0.76

Arg Thr Tyr Asn Ser Bsp LYS Glu His Gln

4 d 3 1 ti 7 19 14 12 5

0.58 063 0.64 0.87 0.70 072 072 0.75 078 @79

type

0.48 0.14

0.78 0.41 0.40 0.24 047 @42

J J

i

8 92

9 16 65

t The 11 unexchanged main-chain amide hydrogen atoms in MBCO are shown in bold. *H,O soaking time, months. $ The 12 unexchanged main-chain amide hydrogen atoms in MBO, are those with exchange <@lo. *H,O soaking time, months. 5 The 2 low exchange main-chain amide hydrogen atoms are in peptides Leu72 (E15) and Leul15 (G16). *H,O soaking time, years, 11The other 5 unexchanged amide hydrogen atoms are in peptides His12 (AlO). LyslR (A14). Lys62 (E5), His81 (EF4) and Glu136 (H12). ‘H,O soaking time. months.

the treatment of bulk solvent and bound water molecules. The inclusion of the solvent contribution to the low-angle reflections has led to significant changes in the surface configuration of the protein particularly in the placement of bound water and ions. In oxymyoglobin, the crystals were grown at pH 57 and transferred to a 2H20 buffer at pH 8.4 and soaked for five months at 2O”C, which gave 8% completely unexchanged main-chain (f <&lo) amide H atoms, 21 y. (0.10 0.60) fully exchanged. Of the unexchanged amide groups in OxyMB, only three are in common to the MBCO structure (all of them in the B helix). The biggest difference is in Leu104 (G5), which is completely unexchanged in MBCO and fully exchanged in OxyMB. MetMB soaked in ‘H20 buffer for four months at pH 5.7 and 4”C, clearly depicted 65% of the backbone amide hydrogen atoms as deuterium, 26 o/o as uncertain, and 9% as hydrogen atoms (Schoenborn,

1971). The MetMB has 13 unexchanged amide groups, and only three agree with MBCO, two in the A and one in the B helix. A MetMB crystal grown from ‘Hz0 at pH 56 and stored in the mother liquor for nearly ten years showed only two slowly exchanging amide groups in the residues Leu72 (E15) and Leul15 (G16) (Raghavan & Schoenborn, 1984). Experience with this MBCO refinement showed that the inclusion of solvent, particularly a shell solvent model, changed the Fourier representation of the surface configuration considerably but had little effect on the details of the internal structure. Therefore, comparison of water sites (all of them located on the surface) should be done only for structures that include the low-angle data with an efficient solvent analysis. The same argument applies to comparisons of hydrogen exchange on the surface of the protein. For interior hydrogen atoms, the observed exchange depends, apart. from the structure itself, on the history of crystallization. The exchange is different in crystals grown in ‘H,O

392

X. Gheng and H. P. Schoenhorn

FYgure 5. Neutron density for Lys56 (LX) from a Fourier (W-F’,) with a model suprrimpowd on it. ‘I’hc~r(~art’ 2 dell sit?; peaks for the atoms beyind the (‘” at,om, an indication that thr residue has 2 cwnformat,ions. or soaked in heavy water. It is expected that other conditions like temperature and pH (buffers) affect the degree of exchange. It should be noted that the observed clearly localized water sites are timeaveraged structures. These water molecules are still exchangeable and possibly rotate in a ratchet. fashion but spend most of the time in the same orientation. The deuterium atoms of side-chains that include 0’H, N’H, N’H, and N’H, groups in MIX0 are fully exchanged. (b) Occupancies

and disordered

side-chains

The individual occupancy for every atom, including all hydrogen atoms, was determined at the completion of refinement keeping all the other parameters fixed in their final values. The average occupancy of main-chain atoms, side-chain atoms and the H” atom are O-97( *O-07), O-93( f O-10) and @95( +0.08), respectively. The distribution of the

averaged observed occupancy of side-chain atoms in Figure 4(c) (beyond the (1” atom) is plotted against residue number. Some of the lysine residues and the residues in regions between helices have somewhat lower occupancies that can be explained in terms of some minor disorder of side-chains. The charged groups have lower occupancies t,han nor)polar groups. The t,hree residues showing evidence for two conformations are Lys16 (A14). I,ys56 (116) and Lys78 (WI). Lys56 (116) has two alternative charged-binding sites (Fig. 5) to Asp27 (KS) and GM2 (D2). The disorders at Lys16 (A14) and Lys78 (EFI) probably involve the charge interactions with two carboxylate oxygen atoms at Asp122 (GH4) respectively. There are five and Glu85 (EFS). residues that show more localized disorders; namely, Glu4 (A2), Lys47 (CM). Lys50 (CM), ThrSFi (FGI) and Lys147 (H23). The existence of disorderd residues in mgoglobin was observed previously and reported hv Phillips (1980), who found two alternative conformations for

Neutron Diffraction

393

Study of Carbonmonoxymyoglobin

Table 6 Disordered side-chains

MBCOJ Kuriyan et (~1.

MBCOT This study Residue

Conformation

Number

Type

Region

4 13 16 45 47 50 56 61 75 78 X6 x9 91 95 96 I”;! IPX I3I 147

c:h.l Val LYS Arg L)X Lys LYS

‘4% A-211 Al4 CD3 CD5 (‘D8 D6

IkU

Et

Ile LYS

El8 EFl Fl F4 P6 FG 1 F(:2 GH4 H5 Hi HZ3

LeU

1XU GlIl Thr I+ Asp (iln Met I+%

t The occupancies t ;UI thla atoms in Notice the very low 4 The 4 disord&wi

(Q> 0.83 092 076 0.93 083 0.81 0.69 0.97 090 0.77 097 094 0.86 0.8 1 0.85 0.90 0.90 098 034

Neutron 1981

X-ray 1998

Neutron 1990

Method gear

22.3 183 190 198 21.9 20.0 17.3 164 1.54 20.2 197 17% 21.6 17.2 19.5 26.4 17.6 12.9 21.9

(8)

MBCO$ Hanson et (LI.

MBO,§ Phillips

6,

(B)

1 and 2

Q

(B)

V’

0.55

11.3

045

12.7

J J

051 0.29

0.9 3.5

0.49 0.7 1

0.9 3.6 J

0.58

10.5

042

10.4

0.41

10.5

059

12.4

J J

V’

0.86 @41

55 79

014 0.59

3.6 99

and temperature factors are averages beyond C@for present conformation. a particular conformation had the same variable occupancy, and the temperature R-factors for certain residues. side-chains are marked by the symbol ,/.

four side-chains in oxymyoglobin. Based on early real-space refinement of neutron data, Hanson & Schoenborn (1981) reported two alternative conformations in MBCO. but four different side-chains compared wit’h OxyMR. In the X-ray model of MKCO seven residues had t,wo alternative conformat,ions (Kuriyan et al., 1986). However, there are no residues common to these three reports. The average B-factors and the refined occupancies for the disordered side-chains are given in Table 6. The agreement between the neutron and X-ray studies is poor; notice the unrealistically low B-factors for certain disordered side-chains (Leu61 (E4), He75 (EIS), Met131 (H7)) in the X-ray model of MBCO (Kuriyan et al., 1986). 5. Hydrogen

X-ray 1980

Bonding

The positions of hydrogen atoms, deuterium atoms and surface water molecules can be determined by neutron diffraction, allowing a more detailed examination of the geometry of hydrogen bonding in the protein molecule. This leads to a better understanding of the interaction of the hydrogen-bond donors with acceptors, and of the role of the interaction on the folded three-dimensional conformation of the protein. Based on the analysis of neutron data from three different myoglobin derivatives, we have described stereochemica.1 restraints on hydrogen bonding with proteins (Cheng & Schoenborn, 1991) and water-

v’

factors arr averages beyond Cq8.

related hydrogen bonding (Cheng & Schoenhorn, 1990). Only intra-protein hydrogen-bonding configuration is discussed here. Columns 8 and 9 of Table 4 describe the geometry of intra-prot,ein (main-chain and side-chain) hydrogen bonding: the distance bet.ween the donor and acceptor. the distance bet.ween deuterium and its acceptor, and the angle between the donor-deuterium-acceptor. Table 7 gives mean values for various types of bonds: in helices and in side-chains. To be included as a hydrogen bond, the donor-to-acceptor distance has to be less than 3.5 a and for side-chain hydrogen bonds the angle at the deuterium atom has to be greater than 120”. The variation in the average ‘H . . . 0 and N . 0 distances among the different helices is quite small. The variation in the angle at the deuterium atom is only 8”. with an average N-*H 0 angle of 147”. Hydrogen-bonded sidechains are divided into four groups according to donor atom: N2H, N’H,, N’H, and 02H groups. The N2H group has almost identical averages with main-chain hydrogen bonds, with the average N . 0 and 2H . . 0 distances of 3.2 !I and 2.3 A, respectively. The O’H group has a quite different hydrogen-bonding geometry from the N2H group: thus, the bond lengths 0. .O and 2H. 0 decrease while the 0-‘H . . 0 angle increases. The average 0 . 0 and 2H . . . 0 distances are 2.7 A and 1.8 A, respectively, and the average 0-2H 0 angle is 156”.

Hrlixt 393&W18 :%17+0‘10 3.20 & 0.07 3.28 + 009 3,15*013 307*0.14 :~12*009 3.lifW16 3-22+01.3 3~17fOl4

3.49 3.34 3.3 I 341 343 3Y28 3-33 347 3.4 I 349

‘5. IS I) 15 I’ f:

H f ‘orner .\II (main) Donor.

I3 I74 I61 152 I i3 I73 170 I65 1.57 I73

124

I 36 I32 I47 I32 I30 12.5 13.5 I 35 Id-1

acceptor (A)

hlin

Max

Avr

hIill

Max

Avr

Min

RlHh

( ) -.ivv

SZH N2H VHZ ;J2H3 ,\I1 (side)

2.83 2.48 2.17 2.67 2.17

3.45 3.33 346 2.75 3.46

3.15*0??z 3.02 + 0.38 2.78kO.40 2.71 *003 297fo.34

193 I ,5.i I ,40 I.77 I ,40

2%9 2.51 2.45 I ,86 2.69

228 * 023 2.12*0.41 I-94 + 0.37 I .82 * 0.03 2.09 * 034

I21 I41 I”1 I40 I”1

I ti3 I67 I69 173 IS.5

117+ I4 I:i-“+ll I43Tli I!x* I.5 IlXi I6

.III (protein)

2.17

3.49

3.12+0-22

I .40

2.69

226 + 092

121

17-i

I4i -i_1”

t Based on a cutoff of a maximum $ Based on a cutoff of a maximum

(a) Histidinr

2H

acceptor (4)

Ihrror

donor to acceptor distance of 3.50 A and helix assignment. donor to acceptor distance of 3.50 A and angle at deuterium

protonation

residues

The percentage of protonation of histidine residues (Table 8) was derived from the observed neutron scattering length of the exchangeable hydrogen atom, and is compared to the assigned pK value of Friend & Gurd (1979). Again we note that, the observed actual ‘H,O concentration is 80% (Cheng & Schoenborn, 1990). The percentage of

‘H

0 accrptl)r

atom greater than I20

is given by:

&(%) = [ (6.67 + 3.73)f- 3.74]/ I(6.67 + 3.74)0+X-

3.741,

where f is the fraction of deuterium present. The deprotonation of the distal histidine residue was discussed in section 3. The present refinement and Fourier maps show that His24 (B5) and His1 19

Table 8 Protonation

of hi&dine

residues Friend & (+urd:

This stud) I’K,,, Atom ‘H”*

Residue Number

Region

12 24 36 48 64 81 x2 lx3 95 113 I16 I I9

A10 u5 Cl CD6 E7 EF4 EF5 F8 FG3 G14 G17 GHl

f-factor 0.37

Q("/,)t

&(%I)

Ph’i”,

Formal t,harges only

Including dipole virtual vhnrges

2

89

6.00

$70

6M

99

7% 660 5.00 6.60

i.ii

7Wi

85 3 90

653 +20 6.55

645 4.1 i 6.67

33 0 31

6.60 660 6.00

0.73 1.oo

41§ 0 48 (41 85 100

079 @42 0.40 W62

97 14 9 59

0.36 @57

A35

6.36 ,545

t The percentage of protonation at pH 57 is calculated from the neutron scattering length: Q(%) = 100(104Of-3,74)/4X. The percentage of protonation at pH 57 is 1:PK,.,, the intrinsic pK values; pK l,z, the value of pK at the pH of half-titration. calculated from the Henderson-Hasselbalch equation: Q(%) = lOO(l+ 10”H-pK)-‘, where pK = pK,,,, includes dipole virtual charges. 0 See section 5(a), Histidine residues. 11See section 3(b). Proximal and distal histidine residues.

Neutron Diffraction His

Study of Carbonmonoxymyoglobin

(85)

24

,\/ I/*--\‘I. F

;/ H

'N

e-

'C C 'I

H

N,'

I \

x4

395

D61

\ .

/ His

119

(:GHl) (0)

Figure 6. His24 (B5) and His119 (GHl) are hydrogen bonded to each other. (a) Two hydrogen/deuterium atoms were pushed away from their suitable places during refinement and replaced by one shared hydrogen atom. (b) Another hydrogen bond is formed between the ‘Hd’ atom of peptide Bl. (GH 1) are hydrogen bonded to each other (share a hydrogen atom, Fig. 6) and confirms the n.m.r. observation reported by Dalvit & Wright (1987). The HE2 atom of His1 19 (GHl) has an occupancy of rig%, and the HE2 atom of His24 (B5) 41%. The hydrogen bond has an NE2 . . . NE2 distance of 2+%i A, an NE2 DE2 distance of 1.93 A and an angle at the deuterium atom of 153”. The early realspace study of CO-myoglobin indicated that His24 is fully protonated and His119 deprotonated (Hanson & Schoenborn, 1981). His119 was assigned a pK value of 600 (which is 67 o/o protonated at

pH 5.7) and His24 was buried and masked in deprotonated form as described by Friend & Gurd (1979). Four hydrogen bonds connect the B helix (low (II)) and GH corner (high (I?)). The hydrogen bond between the D” atom of His24 (B5) and the carbonyl oxygen atom of peptide Bl helps to hold the histidine ring rigidly in place, and stabilize the GH corner with a hydrogen bond between His24 (B5) and His119 (GHl) (Fig. 6). A water bridge is formed between the Dd’ atom of His119 (GHl) and the carboxyl group of Asp122 (GH4). His82 (EF5) and His97 (FG3) are hydrogen

396

X. Cheng and B. I’. 8’chornhorn -

Table 9 Electrostatic

A Inter-elemmtal

pairs

(element:

helical

or non-halical

cstahiliaation

GlU4 (:I116 (:1111X

06’ 08’ Or2 0’2 or2 0” 06’ 06* oc2 (P’ w od2’ oa2 oa2 OE’

.4spdO .4spd7 (:lu41 Glu.52 GlU.54 MU59 Asp60 Asp60 Clu83 Asp122 Asp126 Asp141 Asp141 Mu148

oa3* ()a*

HfXle

s

HWIU?

NC2

s

Ion 174

IS. Intm-elrmentnl o*’ 0” OQ ( P2

pairs

(rlemrnt:

h&-al

.4i? A4

N; N;

4 I6 I31 l38 (‘6 D2 I)4 F,2 E3 E3 EF6 GH4 H2 H17 H17 H24

NC Y2 SC s: S’ S’ s / Y’ yv* s NC N P s SC

OT non-hrlical

N;

( ‘I):! IL? EP

S” NC N’

Asp60 (:lu85 GlulOF, (:lu136 Asp141 (:lul48

I33 EFX Gti HI:! HIT HZ4

N’ NC NC Y2

t (. Inter-protein parr,s 0’2 (:lu18 (:lu38 (Y’ 0” Glu41 (:I&52 or2 OL’ OE2 Glu109 OC’. w* (:lu109 Glu148 OF2

.4 16 ( “3 (ii D2 (:I() GIO H24

0” OE2

p,nir.~

I+79

El3

Lysl33 l+‘ii ArgllX Lys.56 Lys4T Lys34 lJJ%50 Ion209 Arg45 Arg4.5 Vail Lysl6 Ion178 His82 1011218 LysH7 I+87 Arg4.5 lot1283 His97 lJ,VSII.5

H9 El0 c:19 I)6 (‘I).5 ISI5 (‘1)X N2H+ (!I)3 ‘l (‘IN SAl Al4 N2H; EP5 N2H: F” F2 ( ’ I)3 N’H; FG3 H%l

Lys47 Lysf,6 Ly5ti3 Lys63 Lys78 LyslO2 Argl39 Lysl40 I‘m 14.5

(‘IA5 IHi E5 mi F1F 1 (:3 HI5 H16 H21

LJW50

(‘I)8 EF:! El0 (:I)6 FG2 H21 G3’

reyion)

Asp44 (:lu.52 (:lu59

(Y’. OE2 OC’. oc2 oc2

chwgr

wyion)

or* OL’ 0c*

so:~

thrvugh

bonded to the carboxylic acid group of Asp141 (H17) and to the propionic acid group of the pyrrole ring C in the heme. These charged sites play an important role in maintaining the native structure of the protein: they stabilize the highly mobile EF and FG corners and provide a polar interface to form strong hydrogen bonds to water. The His N”* atom close to a carboxylate oxygen atom (which is negatively charged) may explain the high percentage of protonation (positive charge) of these two histidine residues. The geometries of those charged sites differ from the assignments of masked sites for His82 and His97 reported by Friend & Gurd (1979). The NE2 atoms of His12 (AlO) and His36 (Cl) are deprotonated. A ring of four water molecules sits above the plane of His12 (AlO), with an approach distance from -3-2 A to -3.7 A. This deprotonated imidazole ring forms a hydrogen bond to water similar to the kind of interaction suggested by

Lys79 Lys77 His48 Lys96 Lys 147 L-s 102

Levitt 6t Perutz (1988) where aromatic rings act as hydrogen-bond acceptors (Burley & I’etjsko. 19%; Singh & Thornton, 1985). The aromatic ring of Phe106 (G7) and the imidazole ring of His36 (Cl) are parallel and separated by 3.5 8. The imidazolc ring is deprotonated and differs from the abnormal high pK value given to this residue (Friend & Gurd, 1979; see Table 8). Seven histidine residues are hydrogen bonded to water and five form inter-molar hydrogen bonds. Three histidine residues in the inter-helical regions (EF, FG and GH corners) are involved in intraprotein hydrogen bonding. (b) Arg45 X-ray arginine involved

(C’LU)

crystallographic evidence suggests that 45 in sperm whale CO-myoglobin is in ligand access to the heme pocket

Neutron Dilraction

Study of Carbonmonoxymyoglobin

(Kuriyan et al., 1986). The side-chain of Arg45 (03) in MBCO exhibits strong hydrogen-bonding interactions with surrounding atoms in the protein and the solvent. The NE-‘HE group forms a hydrogen bond to the carboxylate oxygen atom 0”’ of Asp60 (E3); N”2-2HV21 forms a hydrogen bond to the Oa2 atom of Asp60 (E3); and NV’-2HV11 forms a hydrogen bond to the propionic acid group. The three hydrogen bonds link three different structural segments. the E helix, the CD corner and the heme. Further, a water molecule links the side-chains of the distal hi&dine residue and Arg45: another water molecule is bound to the main-chain carbonyl oxygen atom of Arg45 and the propionic acid group. This complex hydrogen-bonded linkage of Arg45 to neighboring atoms possibly controls ligand entr? and ligand affinity. The detailed geometries of the hydrogen bonds formed by t’he hydroxyl group (OH) of serine, threonine and tyrosine, and carboxyl group (C=O) of asparagine and glutamine, are listed in Table 4, column 9.

6. Electrostatic Stabilization All water molecules bound to CO-myoglobin are hydrogen bonded to polar or charged groups. Earlier, we gave detailed analyses of the hydrophilic side-chains that form hydrogen bonds with water molecules (Cheng & Schoenborn, 1990). In this section. intra and inter-protein electrostatic intera&ions are discussed. The amino acid side-chains in a protein are categorized as charged, polar or nonpolar. The MBCO protein at pH 57 has 29 positive charges (Fe of the heme not included), and 24 negative charges. The charge status at this pH for individual side-chains are based on the assignment of pK values by Friend & Gurd (1979a,b), except for histidine residues (Table 8). In this analysis, four ammonium ions and one sulfate ion have been located, leaving a net unbalanced charge of + 7. The existence of other bound SOi- is still uncertain and must wait until the analysis of the equivalent nondenatured (H,O) crystal is complete to clearly distinguish between partially occupied water sites and sulfat,e ions. With the measurement of the crystal‘s mass density, the evaluation of the scattering density of solvent shows that ionic components of seven ammonium sulfate molecules per protein are present in the solvent space of myoglobin crystals (Schoenborn, 1988). However, the total of seven ammonium sulfate molecules is generally larger than the number of ions observed in crystal structure determinations. A detailed assessment, of the solvation of ions in protein crystals has not yel been possible and it remains to be demonstrated if local electrostatic effects are strong enough to localize all ions at specific sites on the protein surface or if some fully solvated ions are “free” in the solvent space. Four ammonium ions and one sulfate ion are bound in CO-myoglobin 1990), two sulfate ions in (Cheng & Schoenborn, metmyoglobin (Takano, 1977a,b), and one sulfat,e

397

ion in oxymgoglobin (Phillips, 1984). More sulfate ions have been found in neutron data from metmyoglobin crystals soaked in 4044 2H20 mother liquor (Cheng, 1989). The contribution of electrostatic free energy to the stability of metmyoglobin has been studied by Garcia-Moreno et al. (1985). The relationship between the three-dimensional structure of proteins and electrostatics in proteins has been reviewed by Honig et al. (1986). The behavior of charged residues in proteins can be assessed according to their site specificity. Table 9 lists the individual charged-pair partners at pH 5.7 in CO-myoglobin, the separation between paired atoms, and the regions of the protein molecule involved in the specified stabilizing interactions according to the specific secondary structural motifs. e.g. helical and non-helical elements in glohular protein. lnterartions are classified into intra-protein and inter-protein. There are two different types of intra-protein interaction: interaction within helical or non-helical regions and int,eraction between these elements. The element here refers to the helical and non-helical regions in this globular protein. Intra-elemental electrostatics contribute to the stability of the individual elements of secondary structure, and possibly, to the formation of nucleation units during folding. Inter-elemental electrostatic interactions anchor the structural elements and, thereby, help t.o stabilize the three-dimensional structure of the protein. There are a large number of inter-elemental pairs for the two long helices A and H (Table IO). The A and B helices are the only long helices that have no (01 weak) intra-helical electrostat’ir interactions, hut thev both have considerable inter-elemental ones, particularly the A helix. The long H helix has both large intra and inter-elemental interact,ions. Tn t,he case of the F helix and the FG corner, t’he absence of intra-elemental electrostat.ic intera&ms might be of functional significance: the F helix includes a unique form of inter-elemental stahilization. for it is co-ordinated through the proximal histidine residue (F8) to the heme iron; the FG corner also includes an inter-elemental electrostatic interaction t,hrough His97 to one of the propionic acid groups of the heme. It is significant. that charged atoms t,hat belong to the inter-helical elements are involved in some of t.he strongest specific pairwise interactions throughout the molecule. Three examples are Lys79 (EF corner) with Glu4 (A helix), Asp122 (GH corner) with Lysl6 (A helix), and Arg45 (Cl) corner) with Asp60 (E helix), which are observed at a distance separation of 3.10 A, 2.17 A and 2.99 A, respectively. The ion pairs present in CO-myoglohin show qualitatively that approximately 3O’?;, are intraelemental and 7O0/0 are inter-element,al. The nature of the intra-elemental interactions must, enhance the stability of the secondary structural elements, and the majority of the pairwise int.eractions encount,ered in CO-myoglobin that det,erminr it.s tertiary structure are inter-element,al. Three side-chains of Lys residuras parficipa,tr in

t Element: helical or non-helical

region.

intra and inter-protein electrostatic interactions: Lys77 (E20) sits between Glu18 (Al6) and symmetry-related Glu41 (C6); Lys79 (EF2) between Glu4 (A2) and symmetry-related Glu38 ((‘3); Lys102 (G3) between Glu85 (EFS) and symmetry-related Glu148 (H24). Lys96 (FG2): Glu109 (GlO), and Lys147 (H23) form two charge bridges between three protein molecules. The side-chain of Lys56 (D6) Shows clear evidence for two conformations, as seen in the neutron map (2&,-Fo,) (Fig. 5). In one of these conformations the N’H; charged group is paired with Asp27 (BS) (inter-helical). The alternative position is that the N2H; group shifts to the space nearby Glu52 (D2) (intra-helical). The relative occupancy between the two sites is - 527; and -480/6. The side-chain is disordered only beyond the C6 atom. The two conformations of Lys56 (D6) may also relate to the two conformations of the CO ligand. Examination of the neutron Fourier map of the entire surface boundary region of the protein indicates that there are four cation and one anionbinding sites. Glu59 (E2). Asp126 (H2). Asp141 (H17). and the propionic acid components of the heme moiety show cation-binding regions, while Lys145 (H21) is bound to an anion. Two cation and one anion sites are related to the longest helix H. The cation site bound to the carboxylat’e group of Glu59 (E2) and the cation site close to the propionic acid of the heme, combined with the residues of Xrg45 (CD3) and distal His64 (E7). probably are related to specific mechanisms for ligand entry and binding. Lys42 ((17) and Lys98 (FG4) are observed at a distance separation of 6.0 A, suggesting that there is a potential anion-binding site; which. if occupied, would counterbalance the effect of the destabilizing interaction. The neutron density in this region is much higher than for a sulfate ion, suggesting that there are three 2H20 molecules or a combination of

water and sulfate ions connecting these two lgsine residues.

the side-chains

of

7. Conclusions This neutron diffraction study of CO-myoglobin has shown that the overall fit to t’he observed data is greatly improved, and a final R-factor of 11.3’!/, is achieved by using a modified PROLSQ with a solvent analysis technique. The accuracy of the atomic co-ordinates is improved, with uniform overall occupancy factor for the structure, especially for the side-chains on surface residues of the protein. The solvent structure, composed of water and ions, is better described. Ligand CO binding to heme exists in two nearly equally occupied conformations. The distortion from the linear perpendicular binding geometry is a consequence of steric hindrance due to His64 (E7). Va168 (Ell) and Phe43 (CDI). These two CO locations (42o/o and 58%) may be coupled with other alternative residues that have similar partial occur panty; for example, the two conformations of sidechain of Lys56 (D6) (48 “/ and 52 y/b) and the h?drogen atom shared between His24 (B5) and H1sl19 (GHI) (41 y/O and 59%). The iron is in the least-squares plane of the heme. as observed earlier by Hanson & Schoenborn (1981) and by Kuriyan et al. (1986). One of the two propionic acid groups folds back into the protein t,o form two hydrogen bonds with Ser92 (E7) and His97 (FG3), while another group extends into the solvent region. The co-ordinates of the MBCO protein and water structure have been deposited at the Brookhaven Protein nata Bank, accession number 2 MBfi. The original MBCO data were collected in collaboration with A. C. Nunes and .J. C. Norvell. The original myoglobin co-ordinates were provided by J. C. Kendrew and H. C. Watson. We thank Dr A. D. Woodhead for critically reading the manuscript.

Neutron D$fraction

This research was done under the auspices of the Office of Health and Environmental Research, and calculations were performed under the supercomputing program of the I.J.S. Drpartment of Energy. References Bianconi, A.. Congiu-Castellano, A., Durham, P. J., Hasmain, S. S. & Phillips, S. E. V. (1985). Nature (London), 3 18. 685-687. Brown, W. E., Sutcliffe, J. W. & Pulsinelli, P. D. (1983). Biochemistry, 22, 2914-2923. Burley, S. K. & Petsko, G. A. (1985). Science, 229, 23-28. Cheng. X. (1989). Ph.D. thesis. State University of New York, Stony Brook, NY. Cheng. X. & Schoenborn, B. P. (1990). Acta Crystal&r. sect. R, 46> 195-208. Cheng. X. & Schoenbom, U. P. (1991). Acta. Crystallogr. In the press. Dalvit, C. & Wright). P. E. (1987). J. Mol. Biol. 194, 313-327. Derewenda. Z., Dodson. G.; Emsley, P., Harris, D., Nagai. K., Perutz, M. & Renaud,
Hanson. .J. C. & Schoenborn, B. P. (1981). J. &fol. Biol. 153, 117-146. Hendrickson. W. X. (1985). Methods Enzymol. 115, 252-270. Honig, R. H.. Hubbell, W. L. & Flewelling, R. F. (1986). Annu. Rev. Biophys. Biophys. Chem. 15, 163-193. Johnson, K. A., Olson, J. S. & Phillips, G. N., Jr (1989). ,I. Mol. Biol. 207. 459-463.

Edited

3!N

Study qf Carbonmonoxymyoglobir~

Jones. T. A. (1982). In Computational Crystallography, pp. 3303-3317, Clarendon Press, Oxford. Konnert. .J. H. (1976). Acta Cry.stallogr. sect. A, 32. 614-617.

Konnert,. J. H. & Hendrickson. W. A. (1980) Acta (‘rystallogr. sect. A, 36, 344-349. Kuriyan, J.. Wilz, S., Karplus, M. & I’etsko, (2. A. (1986). .J. Mol. Biol. 192, 133-154. Levitt, M. & Perutz. M. F. (1988). J. Mol. Bid. 201. 751-754. Makinen. M. W.. Houtchens. R. A. & Caughey, W. S. ( 1979). I’roc. Nat. Acad. Rci., C.S.A. 76, 6042.-6046. Norvell. ,J. C., Nunes. A. C. & Schoenborn. R. P. (1975). Science, 190, 568-570. Peng, S. M. & Ibers, ,J. A. (1976). J. ilmer. Phenc. Sot. 98. 8032%8036. Phillips, S. E. V. (1980). J. Mol. Biol. 142, 531 -554. Phillips, S. E. 1'. (1984). In Neutrons in Biology, Basic Life Sciences (Schoenborn, B. P.. ed.). vol. 27, pp. 305-322. Plenum, New York. Phillips. S. E. V. & Schoenborn. B. P. (1981). Nature (London), 292. 81-82. Powers, L., Sessler. ,J. L., Woolery, G. L. 8 (‘hance. B. (1984). Biochemistry, 23, 5519-5523. Raghavan, N. V. & Schoenborn, B. P. (1984). In IVeutrpns in Biology, Basic Life Sciences (Schoenborn. B. I’.. rd.), vol. 27. pp. 247-259, Plenum. New York. Schoenborn. B. I'. (1971). Cold Spring Harbor Symp. &ant. Biol. 36, 569-575. Schoenborn. B. P. (1988). J. Mol. Biol. 201. 741&749. Singh, ,J. & Thornton. J. M. (1985). FEB8 Letters, 191. l-6.

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by B. W. Matthews