Rend. Fis. Acc. D)wei s. 9, v. 4:65-73 (1993)

Biofisica. - - Molecular bases for heme:ligand recognition in sperm whale (Physeter catodon) and Aplysia limacina myoglobin. Nota di M~mco Rtzzi, PAOLO ASCENZI, ALESS~'aDRO CODA, MAumzlo BRUNOV~ e Mamn_NO BOLOGNZSI, presentata(*) dal Corrisp. M. Brunori.

ABs~c r . - - The crystal structure of sperm whale myoglobin (Mb):azide complex has been determined and refined at 1.8/~ resolution. The ligand is coordinated to the heme iron, and deeply buried in the inner part of the distal site. The structural organization of this complex differs substantially from that observed in A. limacina Mb: azide complex, in which the iron coordinated ligand is directed towards the outer solvent region of the protein. In the case of sperm whale Mb the bound ligand is stabilized by a hydrogen bond to residue HisE7; in the case ofA. limacina Mb the ligand is directly hydrogen bonded to ArgE10. The structures of the complexes described underline different mechanisms of ligand recognition and stabilization in the two Mb's, and allow a rational interpretation of the different kinetic and thermodynamic properties observed in globins lacking a polar residue at the distal E7 site.

KEY WOI~DS: Myoglobin; Heme proteins; Biocrystallography; Protein:ligand interactions; Protein structure.

RtaSSUNVO. - - Bad molecolan del riconoscimento eme:leganti nella rnioglob~)~a di capodoglio (Physeter catodon) e di Aplysia limacina. La struttura cristallina del complesso formato dalla mioglobina (Mb) di capodoglio e dall'azide stata determinata a 1.8/k di risoluzione. 1] legante risulta coordinato al ferro eminico, e profondamente sepolto nella parte pith interna del sito distale. L'organizzazione strutturale di questo complesso differisce da quella deU'analogo addotto formato dalla Mb di A. limacina e dall'azide in cui il legante ~ orientato verso l'estemo del sito distale della proteina. Nel caso della Mb di capodoglio il legante ~ stabilizzato da un legame idrogeno con il residuo HisE7; nel caso della Mb diA. k'macina, il legante forma un Iegame idrogeno con il residuo ArgE10. Le strutture dei complessi molecolari descritti indicano l'esistertza di due diversi meccanismi di riconoscimento e stabilizzazione del legante neUe due Mb, e permettono di interpretare le diverse pmpriet~ termodinamiche e cinetiche osservate nelle globine prive di un residuo polare al sito distale E7.

INTRODUCTION

Oxygen-carrying heme proteins transfer or store molecular oxygen in several phyla among vertebrates and invertebrates. They share a highly conserved three-dimensional structure (known as the globin fold [1]) which is little affected in its spatial organization despite low sequence homology, and overall assembly (whether monomeric or oligo- meric) [2-4]. The mechanism of ligand stabilization in the distal site of heme proteins has been studied thoroughly, not only for oxygen, but also for anionic ligands of the ferric form. In the course of our studies on the functionality of lower phyla rnonomeric globins, we have become interested in the stuctural and functional properties of the distal site of globins lacking the distal histidine, HisE7 (1), shown to stabilize through direct hydrogen

(*) NeUa seduta del 9 maggio 1992. (1) Residues of sperm whale and A. limacina Mb have been identified by their topological positions as

defined by Perutz [1, 3]. The eight helices in the globin fold are identified by letters A,B,...H. The symbols AB, BC ..... GH, indicate the nonhelical regions connecting helices A and B, B and C, ..., G and H. Numbering and nomenclature of the heme group are those adopted by Takano [5]. Water molecules (W) have been numbered sequentially, starting from 150. Abbreviations used: myoglobin, Mb; monomeric hemoglobin, Hb; monomeric erythrocruorin, Ery.

66 M. RIZZI ET AL.

bonding the ligand coordinated to the heme iron [5, 6]. In the case of A. limacina Mb, the lack of a polar residue at position E7, is compensated by the presence of a long and flexible Arg side chain at position El0. Upon ligand binding ArgE10 folds into the distal site and triggers the formation of extended polar interactions which comprise the ligand itself, ordered water molecules in its neighborhood, heme propionates and protein atoms from the CD region of A. limacina Mb [7-9]. Thus, despite the substantially different ligand stabilization mechanisms adopted by proteins displaying very similar tertiary structures, the overall binding free energy of some selected ligands to globins can be quite comparable far apart on the evolutionary scale.

Our recent crystallographic investigations on the binding mode of azide to A. limacina Mb have shown that interaction with ArgE10 forces the iron-bound linear anion in an orientation protruding towards the solvent side of the distal pocket [7]. On the other hand, a preliminary structural report on the crystal structure of the sperm whale Mb:azide complex [10] indicated that in this protein azide bound to the heme iron is deeply buried in the distal site crevice, pointing towards the G heLix inner face. In order to firmly establish the heme:ligand orientation and its significance in the different ligand stabilization mechanisms observed for the two Mb's, we have carried over the crystallographic refinement at 1.8/~ of the sperm whale Mb:azide complex, which is described in this report, in parallel with a discussion on related functional data for different monomeric globins.

M A T E R I A L S A N D M E T H O D S

Aquo-met sperm whale Mb crystals were prepared from commercial protein preparations (from Sigma Chemical Co., St. Louis, MO, USA) as described by Bolognesi et al. [11]. All the chemicals, from Merck AG (Darmstadt, Germany), were of analytical grade, and used without further purification. In order to prepare the samples for X-ray data collection the crystals of the aquo-met form of the protein were soaked in 0.05 M phosphate buffer solutions containing 3.2 M ammonium sulfate and 0.01 M sodium azide, pH 7.0, at room temperature for 48 hours. They were subsequently sealed in glass X-ray capillaries for the diffraction experiments. One crystal allowed for the collection of a full data set of 1.8.31 resolution, using a conventional sealed tube CAD4 diffractometer (ENRAF-NONIUS, Delft, The Netherlands), equipped with a helium flushed extended detector arm (368 mm). Data reduction, which included correction for the decay of the crystal (maximum intensity decay 19.2%), and for crystal/capillary absorption, was performed with the standard ENRAF-NONIUS SDP program package. A total of 11.712 intensities (with I > 1.0or(I)) were collected and subsequently reduced to structure factors, corre, sponding to 92% completeness of the 11.0-1.8 ]i resolution shell. Starting Fourier maps were computed with coefficients 2Fd-Fn, Fd-Fn, and phases calculated on the basis of the sperm whale Mb aquo-met crystal structure deposited with the Brookhaven Protein Data Bank (Fd and Fn are the moduli of the observed Mb:azide complex and of the calculated aquo-met Mb derivative structure factors, respecti- vely) [5, 12]. The side chains of residues ArgCD3, AspE3, and HisE7 which could have

MOLECULAR BASES FOR HEME:LIGAND RECOGNITION .. . 67

varied their conformations upon ligand association, were purposedly omitted from starting phase calculations. The crystallographic R-factor calculated at this stage was 25.9%, based on the 11.0-1.8/~ resolution data set.

Inspection of the starting maps showed prominent electron density peaks at the distal site compatible with the bound anion and with the side chain of HisE7, in its ~<closed gate~) conformation. Moreover, density for the other omitted side chains was present. In order to avoid bias resulting from the calculated phases, only the atomic structure of the azide anion was fitted to its observed electron density in the distal site, at this stage, and conventional restrained crystallographic refinement was undertaken. The program package TNT [13], together with the molecular graphics program FRODO [14] were used throughout. The side chains of ArgCD3, AspE3, HisE7 were introduced into the model, and in phase calculation, following inspection of the difference Fourier maps calculated at the end of the first refinement cycle (R-factor 17.3%), which confirmed that these residues adopted a conformation quite close to that observed in the starting model coordinates. After a total of 4 cycles of refinement/model building the crystallographic R- factor, calculated in the 11.0-1.8/~ resolution range, was 14.9%. The r.m.s, deviation from ideal bond lengths in the model is 0.016_~, r.m.s.; deviation from ideal angles is 2.43 ~ . The model included 123 ordered solvent molecules, for which occupancies were refined in the final stage of the analysis. Independent crystallographic refinement of the azide ligand occupancy showed that in the crystal the heine distal binding site is fully occupied by the anion, as also shown by microspectrophotometry on single crystals (A. Merli, unpublished results). The final refined coordinates for the sperm whale Mb:azide complex have been deposited with the Brookhaven Protein Data Bank [12], from which copies are available.

R E S U L T S A N D D I S C U S S I O N

Inspection of the initial difference Fourier maps indicated immediately a substantial agreement of the structure of the sperm whale Mb:azide complex determined by us with that proposed by Stryer et al. [10] on the basis of a 2.0/k difference Fourier map, calculated with multiple isomorphous replacement phases. As shown in figs. 1, 2, after crystallographic refinement, the linear anion is found deeply buried in the distal crevice of sperm whale Mb, coordinating to the heme iron by means of the N3 atom. The azide molecule is oriented along a direction which, once projected on the heme piane and starttng from the iron, points to the center of the 2C4-ACH heme bond (ACH is the methinic carbon more deeply buried in the heme crevice).

Upon ligand binding the distal histidine, which may transiently swing to an ~opem) conformation with respect to its position in the aquomet protein in order to leave room for the incoming azide, adopts a <<closed~) conformation, shielding the bound anion from solvent contact. Moreover, the HisE7 Ne atom is at hydrogen bonding distance from the azide atom N3, which, in turn, is coordinated to the iron. In this orientation the conformation of HisE7 is virtually coincident with that adopted by the same residue in the aquo-met form of the protein in the presence of H20 as a ligand. As shown in figs. 1, 2,

68 M. R / Z Z I E T A.L.

Fig. 1. - Stereo view of the distal site of the sperm whale Mb:azide complex, determined at 1.8 ~ resolution.

"•ArgCD3 N

Heme • ..- N ...... O I1]: O ..'3o2~,,,:: - -

propionate ])---O" W 218 ' b ~ Asp E3

I j, 2.~8 X ~'"o'" N \5 2.8~ . . ! " 'W~45 N1 -(" / W192~: .... ! ___~..~.N/~ ,~/ /

~ N F I 1N32.91~ "~ V L y s E 6 / o ,

2~. A~ ~ ~ , , E helix o

Heine propionate I g

Fig. 2. - Skematic view of the distal site in sperm whale Mb:azide complex, as depicted in fig. 1. Atom N1 of azide is in the background of the picture. The average Fe...N(pyrrole) distance in the heme is 2.00/~, the iron laying 0.06/~ out of the four pyrrole N plane toward HisF8. The Fe-N3-N1 angle is 117 ~ and the axial

coordination bonds form an angle N8(F8)-Fe-N3 of 179 ~

M O L E C U L A R B A S E S F O R H E M E : L I G A N D R E C O G N I T I O N . . . 69

no other direct polar interaction is found to occur between azide and the protein. Three ordered solvent molecules (W192, W218, W245), showing temperature factors compa- rable with those of the surrounding protein atoms, contribute to the structure of the near environment of the ligand in the distal site, and to the conformation of the heine propionates. These water molecules in the outer region of the heme crevice are not present in the crystal structure of the aquo-met form of the protein, but do not affect the conformations of ArgCD3 and AspE3, which are involved in electrostatic interactions extending from the heme propionate IT[ (upper in figs. 1, 2) to the side chain of residue LysE6. Their presence, as in the case ofA. limacina Bib (see figs. 3, 4) may be related to the stabilization of the distal site structure through extended &localization of the polar interactions taking place in the presence of the anionic ligand.

Inspection of other regions of the sperm whale Mb molecule, not directly involved in ligand binding, shows only minor conformational readjustments on some solvent exposed lysine side chains with temperature factors well above the average for the whole structure. Therefore they do not appear to be a significant feature of this particular ligated state of the molecule. Of the two sulfate ions present in the structure of sperm whale Mb aquo- met form, none is fully retained in the Mb:azide complex crystalline lattice. Inspection of the difference Fourier maps indicates that only one sulfate anion is present per molecule, in a new site, at hydrogen bonding distance from the peptide nitrogen of residue AspE3. Changes in the positions of these interstitial anions may be in keeping with the damaging effects of higher azide concentrations on the stability of the crystals, as observed in the preliminary soaking experiments. The average temperature factor for the 123 located solvent molecules is 34.5 ]k 2, whereas the protein itself shows an average B value of 16.7 ,~2 for the main chain atoms (including CB atoms) and of 29.5 ~2 for the side chain components.

The binding modes of azide to sperm whale and to A. limacina Mb's can be compared at atomic resolution on highly refined crystal structures, in order to correlate this information with existing functional data [4, 7, 15-17] (see table I). In sperm whale Mb, a stabilization of the ligand is provided by the side chain of residue HisE7, which can hydrogen bond to azide with its Ne atom. This favours an orientation of the azide towards the back of the distal crevice (pointing towards residue IleG8), burying a negatively charged species in a protein environment which is apolar but electrostatica]]y positive, because of HisE7 side chain and of the contribution of the ferric iron in the heme. On the other hand, model building shows that an orientation of azide towards the solvent side of the distal crevice (as in A. limacina Mb) would force HisE7 side chain in quite an ~opem) conformation [18], not as productive in terms of ligand stabilization through hydrogen bond, steric collisions of the HisE7 side chain, and in terms of ligand shielding from interactions with the solvent. It is plausible that, because of altered dielectric constant in this more open environment, this alternative orientation of azide in sperm whale Mb, be also unfavoured on the basis of less favourable elecrostatic contributions.

From inspection of the kinetic rate constants reported in table I, two different trends become evident, which can be qualitatively related to the known structural properties of

7 0 M. RIZZI ET AL.

Fig. 3. - Stereo view of the distal site of the A limacina Mb:azide complex determined at 13 ,~ resolution.

PheC7

~ C / A laCD2 I I 2"93 '~ ~ / . . . . O. ~ 2.81A .N 2.99 J,\ A s p u u , : i ,̂ , o;,';?a~'""" {~28ax N.. j

. . . . . w " t . . ' " T H e m e 3.17J, ' -~ ' / 2 . 6 6 ~ t ~ . ' J " : 2 7 7 / ~ ) 2 . 6 4

0 ......... ~--..".. F~__./ l propionate I I I W 2 3 9 .'-' \ _ v . N

~ l JL280 .-" 0"" 6 ~""-. :."2 8, ~ ?

ol 2 0 4 J l N3 279X" \ / / . . . . . �9 . ".. " y " / L y S I : J /

\ HisF8 i (" ~ / O0 ~ ~ e l i x

Heme prop ionate IV

Fig. 4. - Skematic view of the distal site "in A. limacina Mb:azide complex, as depicted in fig. 3. Atom N1 of azide is in the foreground of the picture. Atom NE2 of ArgE 10 is directly hydrogen bonded to N 1, as indicated by the dotted line. The average Fe...N(pyrrole) distance is 1.97 ~, the iron laying 0.08 ~ out of the four pyrrole N plane toward H.isF8. The Fe-N3-NI angle is 122.6 ~ and the axial coordination bonds form an angle Ne(F8)-

Fe-N3 of 169 ~

MOLECULAR BASES FOR HEME:LIGAND RECOGNITION ... 71

the distal sites of the different globins listed. On one side, the association rate constants increase in the series sperm whale Mb -+ Glycera dibranchfata Hb; on the other, azide dissociation rate constants decrease in the series G. dibranchiata Hb -~ sperm whale Mb. As a result of the two opposing processes maximum affinity for the azide ligand is reached in the case of Chironomus thummi thummi Ery (type II).

TABLE I. -- Values of kinetic and thermodynamic parameters for azide binding to monomeric heine proteins.

Posit ions/Residues

H e m e protein E7 E l 0 k on k off K eq ( M - i s - l ) ( s - l ) (M - l )

Sperm whale M b (a) His Thr 2.5 x 10 4 0.9 3.4 x 1.0 4

C thummi thummi Ery (s) His Arg 1.8 x 10 6 2.0 8.5 x 10 ~

A. limacina Mb (c) Val Arg 1.8 x 10 6 7.5 x 10 2 3.1 x 10 ~

G. dibranchiata H b (a) Leu Lys > 3.7 x 10 7 > I x 10 5 3.7 x 10 2

if) pH=6.0, and 25 ~ [4]. (b) pH=6.1, and 25 ~ [17]. if) pH=6.1, and 25 ~ [16]. (J) pH=7.0, and 20 ~ [7, 15].

Control of the association rate constant in the proteins listed in table I is achieved through enhanced/decreased accessibility of the distal site to the incoming Iigand. Thus, in sperm whale Mb, which displays a iron-bound water molecule, and a <<closed gate>> conformation for HisE7 (stabilizing the coordinated water molecule in the distal site of the resting ferric protein), association of azide requires the removal of these two physical barriers to the incoming ligand, resulting in a slow association rate constant. In C. thummi thummi Ery the HisE7 residue is in an <<open gate>> conformation [19], and the sixth coordination position of the iron is not occupied by a water ligand molecule [19]. As a result the association rate constant for azide is as fast as in the case of A. limacfna Mb, which shows an open distal site, and lacks the iron-bound water molecule. A comparable situation has been observed in G. dibranchiata Hb, which displays at the distal site residue LeuE7 [20]. Absence of a water molecule at the sixth coordination position of the heine iron appears to be a structural feature in proteins which lack a properly positioned polar residue at site E7.

Inspection of the dissociation rate constants listed in table I shows a clearcut distinction between globins capable of direct ligand stabilization through HisE7, and proteins which have to rely on a differently positioned polar residue, such as ArgE10 in the case ofA. limacina Mb. The tight binding mode of azide to sperm whale Mb has been related to a <<closed gate>> conformation of HisE7 stabilizing the bound ligand. On the other hand, recent site directed mutagenesis experiments on sperm whale Mb have shown that an increase in the ligand dissociation rate constant, observed for the I-IisE7 -+ Val mutant, can be substantially counterbalanced by the additional ThrE10 -+ Arg

72 M. RIZZI E T AL.

substitution [21 ]. From a comparison of the kinetic constants, from model building, and from consideration of the crystal structure of the C. thummi thummi Ery:cyanide complex [19], a binding mode, with a HisE7 in the <<closed gate>> conformation, can be proposed also for the Ery:azide complex. Owing to the strong stabilization of the ligand, in both cases a slow dissociation rate constant is expected. On the other hand, the azide stabilization mechanism adopted byA. limacina Mb, through ArgE 10, is not as efficient as that based on HisE7, the anion binds with different geometry, more prone to solvent collisions/exposure, and the dissociation process is faster [7]. The same holds, presumably, for G. dibranchiata Hb, which can achieve only a lower degree of ligand stabilization through residue LysE 10, which offers less hydrogen bonding potentialities than in the case of ArgE 10, and cannot support precise protein:ligand:solvent interactions in the distal site.

The analysis here reported shows that functionally related proteins, which as expected display highly conserved three-dimensional folds, indeed base their mechanism of action on molecular properties which differ substantially in their fine details. We believe that such an observation applies, as a principle of evolutionary biology, to other protein families, for which the existence of a common fold is associated to substantially or completely different tertiary interactions controlling the rate parameters of their biological function.

ACKNOWLEDGEMENTS

We are grateful to Prof. Angelo Merli, University of Parma, for the single crystal microspectrophotometric measurements performed on the sperm whale Mb crystals. This work has been supported by CNR ~<Progetto Finalizzato Biotecnologie e Biostrumentazione>~, <<Progetto Speciale Peptidi Bioattivb,, and by the Ministry of University and Scientific-Technological Research of Italy.

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P. Ascenzi: Dipartimento di Scienza e Tecnologia del Farmaco

Universit/l degli Studi di Torino

Via P. Giuria, 9 - 10125 TovaNo

M. Bolognesi: Gruppo Biostrutture - IST

Universit/* degli Studi di Genova

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M. Brunori: Dipartimento di Scienze Biochimiche <<A. Rossi FaneUi>>

Universit~ degli Studi di Roma <<La Sapienza>>

Piazzale A. Moro, 5 - 00185 ROMA

A. Coda, M. Rizzi:

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Sezione Biologia Molecolare e Biofisica

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