Suggested binding mechanism of the HIV-gp120 to its CD4 receptor

THEO CHEM

ELSEVIER Journal of Molecular Structure (Theochem) 367 (1996) 159-186

Suggested binding mechanism of the HIV-gpl20 to its CD4 receptor

Z&in Sztkelya*b’c9*, Z&tin K6nyad, Attila Becskei a, William P.D. Goldringb, And& PerczelbYeTf, Botond Penke

a, Jdzsef MolnBrg, Christopher F. Michejda’, Adorjan Asza16sh, Imre G. Csizmadiab

‘Department of Medicinal Chemistry, Albert Szent-Gydrgyi Medical University, Dam ter 8,H-6720 Szeged, Hungary

bDepartment of Chemistry, University of Toronto, Toronto, Ont. MSS lA1, Canada

‘Molecular Aspects of Drug Design Section, MSL, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center,

Frederick MD 21702-1201, USA

dDepartment of Applied Chemistry, Attila Jbzsef University Rerrich Bela ter 10, H-6720 Szeged, Hungary

‘Department of Organic Chemistry, Lbrdnd Eiitviis Universi& P&m&y Peter Setany 2, H-1518 Budapest, Hungary

fDepartment of Biochemistry University of Oxford, South Parks Road, Oxford, OX1 3QV, UK

gDepartment of Microbiology, Albert Szent-Gyiirgyi Medical Universiry, Dam ter 10, H-6720 Szeged, Hungary

hCell Biology Laboratories, Food and Drug Administration, Washington DC 20204, USA

Received 20 August 1995; accepted 30 January 1996

Abstract

The molecular recognition and attachment of the CD4 molecule and the HIV envelope glycoprotein (gp120) might be described as a consecutive three-step molecular recognition process. (a) Long range interaction: electrostatic pre-orientation, (b) short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation) and (c) internal interaction (induced fit): conformational readjustment of the protein molecules. On the basis of the preliminary investigations (X-ray structures of CD4 and biological studies of CD4 and gp120 point mutants) we described a computational model. This approach consists of empirical calculations as well as ab initio level of quantum chemistry. The conformational analysis of the wild type and mutant CD4 molecules was supported by molecular mechanics and dynamics (Amber force field). The latter analysis involves the application of a novel method, the Amino Acid Conformation Assignment of Proteins (ACAP) software, developed for the notation of secondary protein structures. According to the cardinal role of the electrostatic factors during this interaction, several ab initio investigations were performed for better understanding of the recognition process on submolecular level. Using the above mentioned computational model, we could interpret the basic behaviours and predict some additional features of CD4-gp120 interaction, in spite of the missing gp120 X-ray structure.

Keywords: Ab initio calculation; Anti-HIV drug; CD4; gp120, Docking; MM and MD simulations

1. Introduction

1.1. Biological background

* Corresponding author. Fax: (l-301) 846-6231; e-mail: szeke- [email protected]

0166-1280/96/$15.00 Published by Elsevier Science B.V. PII SO166-1280(96)04501-O

Acquired immunodeficiency syndrome, AIDS, is caused by a retrovirus known as human immuno-

160 Z. S.z&ely et al./Journal of Molecular Structure (Theochem) 367 (1996) 159-186

Fig. 1. Soluble rat-CD4 showing the Dl, D2, D3 and D4 domains. The carbohydrate moiety is arbitrarily placed at Asn”’ where one of the

carbohydrate antennas of human-CD4 is located.

2. Szikely et al.iJournal of Molecular Structure (Theo&em) 367 (1996) 159-186 161

deficiency virus (HIV). In the infected patients the activity of a subgroup of the peripheral T-lymphocytes decreases. These special T-cells possess a cell membrane surface glycoprotein, called the CD4 receptor [l]. The CD4 molecule, as a membrane protein, serves as specific receptor for the HIV. However, CD4 also serves as a co-receptor for class II MHC (Major Histocompatibilty Complex) proteins which are involved in the antigen presenting process [2]. The CD4 molecule consists of over 400 amino acid residues with two glycosylation sites [3]. The four domains (Dl, D2, D3 and D4) of the extracellular part of CD4 may be treated as two separately folded superdomains (DlD2 and D3D4) since the D2-D3 junction is highly flexible. All four extracellular domains of CD4 were subjected to mutagenesis. The activity of mutants were determined by standard biological techniques [4-121. The structure of the DlD2 superdomain of the wild-type (WT) was 9etermined by z-ray crystallography. Initially a 2.4 A [13] and 2.3 A [14] resolutio&was obtaine! but recently som: improvement was achieved (2.3 A [15] and to 2.2 A atomic resolution 1161). The continuous’ effort to crystallise the human-D3D4 fragment has not yet been honoured. On the other hand, the structure of the rat-D3D4 [17,18] is readily available. Since the rat-CD4 has only a single glycosylation site, in contrast to human-CD4 that has two, it was easier to crystallise. The conformation of a biantennary oligosaccharide chain, that is nearly identical to the human oligosaccharide chain of the CD4, was investigated by NMR in water [19]. On the basis of X-ray and NMR data a schematic three-dimensional structure of the entire CD4 [c.f. Fig. 1.1 was established at the start of this research. The carbohydrate chain has been attached to the Asn”’ residue as this is the conservative glycosylation site of mamalian species.

The numerous CD4 mutants, already investigated, leave shown either a significant drop or practically no change of both types of biologic,al activity (i.e. MHC II and/or gp120 binding). Based on these analyses both the hydrophobic side chain df Phe43 of the C” strand and three positively charged Lys”, Lys& and Arg5’ residues (C’,C” and D strands) (c.f. Fig. 2 and Fig. 3) are likely to contact gp120. These data are also supported by additional measurements performed on the binding ability of CD4 mutant glycoproteins. For

Di

l

b2

Fig. 2. A schematic illustration of the ridges (from A to G) and interconnecting segments of the Dl domain of the CD4. Note that ridges C’, C” and D, as well as their interconnecting segments stick out of the main body of Dl.

example the ALPHA, GAUGE’, Asp4”, Trp427 and Tyr435 residues on the surface of gp120 [20,21] may act as counterparts for those amino acids of CD4 mentioned above. Thus, the formation of three ‘salt bridges’ may be encountered with an ‘aromatic stacking’ between the Phe of CD4 and the Trp as well as the Tyr of gp120 (c.f. Fig. 4 and Fig. 5). The latter interaction between aromatic side chains is unusual for globular proteins, since their surface is typically sheltered by solvating water molecules. Therefore, an apolar side chain, such as a phenyl group would not stick out from the protein unless it has a dedicated role as it is presumed for Phe43 (c.f. Fig. 6). Since the primary purpose is to find connection between the protein structure of the CD4 receptor and its gp120 binding activity (c.f. Fig. 4 and Fig. 5), a number of CD4 mutants had to be investigated where the replacement of amino acids took place at strategic positions.

The initial event, in the HIV infection, involves the interaction of the virus envelope glycoprotein, gp120, with the CD4 receptor, thereby allowing for the virus in the subsequent step to enter into the human T-cell. Since the HIV infection will be effective if and only if

162 Z. S&ely et aWourna1 of Molecular Structure (Theochem) 367 (1996) 159-186

Fig. 3. Cationic side chains (Lys”, Lys& and Arg5’) shown in red, the side chain of .4sn5* shown in blue and the side of Phe43 is shown in yellow.

2. SAely et al,/Journal of Molecular Structure (Theochem) 367 (19%) 159-186 163

lmir acid sidu

P les

CD4

D2

271 CARBOHYrjRATE

D3

m CARBOHYDRATE

9P 120

H c5 v5

331

v3

296

I 511

c2 aa;iiT

restdues

Fig. 4. A hypothetical relative orientation of CD4 and gp120 showing the ionic and aromatic amino acid residues that might be involved in the CD4-gp120 complex formation.

the gp120 of the virus will bind to theCD4 receptor of the human T-lymphocytes, therefore the prevention of such a binding may be regarded as a possible chance for a successful therapeutic intervention.

Consequently, among the different chemotherapeu- tic possibilities the selective inhibition of the virus binding procedure has been investigated [22].

9P 120

Fig. 5. A schematic illustration of the amino acid residues involved of electrostatic preorientation (Al) and electrostatic attachment (B2) as well as aromatic lock of docking (B3) via Phe43.

1.2. Computational background

It was observed in a previous theoretical work that the guanidinium-aspartate pairing is favoured in the ionic form or ‘salt bridge’ [23]. The modelling of the carboxy peptidase A (CPA) binding site [24], with three (3) strategically located guanidinium ions (to mimic Arg’l, Arg’*’ and Arg’45) indicated an ionic type interaction. The spatial approach to its fmal site (Arg145) was also studied and showed that first the format ‘slides’ over two arginine residues (Arg7’ and Arg ‘*?) before reaching the final binding site, Arg 145. As the formate ion displaces toward the final binding site, it maintains two hydrogen bonds to either one or two arginine residues. Other studies [25] of methyl guanidinium acetate binding, using a 6-31G’ size basis set also indicated that an ion-pairing (or a ‘salt bridge’ formation) is favoured. In the lowest energy configurations, where side-on and end-on sym- metrical twin hydrogen bonds are formed, this is in accordance with X-ray data. A comprehensive study of the X-ray structures of 74 proteins [26] indicated that the ion pairing was favoured between guanidine residues and carboxyl groups. In most casesJhe distance between thesepoups yas less than 4.2A with a typical value of 2.6A to 3.OA.

Previous studies [23] of acetate and formate ions binding to methyl ammonium cation indicated that the neutral-neutral hydrogen bonded complexes are ener- getically favoured. The bifurcated structure is of a lower energy over the single hydrogen bonded structure [27]. In addition, the increased methylation of methyl or ethyl ammonium ion successively weak- ened the interaction energy indicating that hydrogen bonding is indeed important. The study of the reversal of charged residues, that is Lys-Asp for Asp-Lys, between enzyme and substrate was found to be unfavoured [28]. This unfavoured interaction was explained by the differing interactions of the micro- environments. In other words, the enzyme likely folds in such a manner so that a given micro-environment of the active site is pre-polarised to stabilise a particular charge and repel the opposite-charge.

2. Scope

The molecular recognition and with the binding

164 2. S&ely et aL/Joumal of Molecular Structure (Theochem) 367 (1996) 159-186

Fig. 6. The spatial orientation of the benzyl group (-CHI-Ph) in Phe” with respect to the rest of the Dl domain. It is quite unusual that an apolar

(hydrophobic) side chain sticks out of the molecule towards the aqueous solution.

Z. S&ely et al.IJournal of Molecular Structure (Theochem) 367 (1996) 159-186 165

LyS-NH,+ -OOC-Asp -_) /H

,_y&,: . . .

‘H

HOOC_Asp

H,

Arg-C +’ NH,

-OOC-Asp --)

’ NH, Arg-C

// N: ......... HOOC-Asp

’ NH,

’ salt-bridge Bwinsted (H-bonded) complex

Scheme 1. Complexation forms between ionic side chains.

mechanism of HIV-gpl20 into its CD4 receptor has been subdivided into two major steps (A,B), followed by a third phase (C) frequently referred to as an internal interaction:

(A). Long range interaction: electrostatic pre- orientation. (B). Short range interaction: electronic attachment followed by a ‘Locking-in’ (via aromatic ring orientation). (C). Internal interaction (induced fit): conformational readjustment of the protein molecules.

It is assumed that the driving force in the early pre- orientational phase (A) is electrostatic. This is due to the attraction between the side chain endings of - NH3’ (Lys35, LYS~~) and -C(NI&)~ (Arg59) of the CD4 and the -OOC-moieties (AMPLY, GAUGE’, Asp457) of the HIV-gp120.

The interaction between the positively charged amino acid side chains of the CD4 and the negatively charged amino acid side chains of the gp120 (Scheme 1) may be salt-bridged or may be involved in hydrogen bonding (Brcdnsted) complex formation (B).

Phase B is due to the interaction between either the (gp12O)-T1p~~~ and the Phe43-(CD4) or the (gp120)- Trp427 and Tyr435 and the Phe43-(CD4).

The formation of these possible Bmnsted or Lewis complexes (Scheme 2) were envisaged to be of following types:

the

(gpl20)-TrpQ7 ._...._. Phe43-(CD4)

I I (gp 120)-Trp427 P;e43 Tyr435-(gp 120)

CD4

Scheme 2. Complexation forms between aromatic side chains.

The scope of the present paper does not include the final phase (C) of binding.

In order to prove this hypothesis, the following investigations were performed in our laboratories:

molecular dynamical (MD) calculations on CD4 (wild-type and mutants), amino acid conformational assignments of MD calculations based on an ab initio model (ACAP), ab initio calculations on suitable models for electrostatic interactions.

3. Methods

The input geometry of the Dl and D2 domain of the CD4 and its fragment were created by the Insight II 2.1.0 program [29], on the basis of the Brookhaven Protein Data Bank file pdblcd4 aad file pdb2cd4 hav- ing an atomic resolution of 2.3 A and 2.2 A, respectively. Hydrogens were added to the heavy atoms automatically according to the AMBER force field [30]. The molecular geometry optimisation and MD simulations were performed using the AMBER force field potential within Discover 2.8 [29] program on an IBM RS6000/320H workstation. Gradient geometry optimisation was performed on both of the crystal structures, while the MD simulation yas carried out on that of the highest resolution (2.2 A) CD4 crystal structure. The following options were used for calculations: (1) a standard charge set from the AMBER “all atom” force field, with (2) distance dependent

Table 1 Average torsional angles (r#&) values of the basic backbone conformation types or ‘conformational centres’ obtained by averaging of For-(Ala)z-NH* computed at the HF/3-21G level of theory

Conformational

types

ffL - 68.6 - 17.5

aLi 61.8 31.9

PL - 167.6 169.9

Yr - 84.5 68.7

Yo 74.3 - 59.5

6, - 126.2 26.5

6, - 179.6 - 43.7

CL - 74.7 167.8

ED 64.7 - 178.3

166 Z. .Wkely et al.fJournal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 2 Conformational assignment of CD4 by X-ray followed by MM geometry optimization. The X-ray disagreements are marked by * and subsequent MM conformational agreements are printed in bold

lcd4 3cd4 No. AA

x-my MM MM x-ray

1 LYS 2 LYS 3 Val 4 Val 5 Lcu 6 GUY 7 LYs 8 LYs 9 GUY

10 Asp 11 Tilr 12 Val 13 Ghl 14 Leu 15 Dir 16 Qs 17 lhr 18 Ala 19 Ser 20 Ghl 21 LYs 22 LYs 23 Ser 24 Ile 25 GlIl 26 Phe 27 His 28 Trp 29 J-Ys 30 Asn 31 Ser 32 Asn 33 Gln 34 Ile 3.5 LYs 36 Ile 37 Lcu 38 GUY 39 Asn 40 GUY 41 GUY 42 Ser 43 Phe 44 Lcu 45 Tbr 46 LYs 47 GUY 48 Pro

bL

QL l 8L *8L *YL 'CL eL CL QD CL fL *CL YL tL CL 8L l YL +YL f L *8L YL *CL YL *YL l YL 8L 8L fL 8L eL *YL *6D *6L YL CL aL 8L BL *YL YL YD 6L *8L tL *YL 'EL *CD YL

a, CL

A

8L

IL

CL fL fL YD CL eL 8L YL tL 6L a, YL

YL

YL Q

YL

CL eL

PL

CL 8L

8L

CL 8L

CL

YL

ID

6L

YL

eL QL

8L 8L YL

YL

‘yo

aL

BL

CL

8L

8L

CL

ffL

bL

CL

8L

8L

eL

CL

Cl.

CL YD CL 8L BL YL

CL fl,

BL YL fL 8L aD YL

8L

TL

eL

(L

8L

8L

8L

8L

fL

w aL

aD YL

CL 01.

8L

8L 8L

IL

YO w. YL

YL

fL

YL

fL eL

a, 9 eL* h’ CL* 8L* CL CL aL 9

eL 8L* YL

tL CL 8L CL* 8L*

EL

aD*

YL

8L* YL

tl*

Iz 8L CL 8L CL aL* aL* aD* YL CL QL 8L 8L CL* YL 'YD fiL CL* CL eL+ YL* CL YL

Table 2 Continued

lcd4 3cd4 No. AA

x-ray MM MM x-my

49 Ser 50 LYs 51 Leu 52 Asn 53 Asp 54 Arg 55 Ala 56 Asp 51 Ser 58 Arg 59 Arg 60 Ser 61 LtU

62 Trp 63 Asp 64 Glu 65 GUY 66 Asn 61 Phe 68 Pm 69 LeU

70 Ile

71 Ile

72 LYs 73 Asn 74 J&J

35 LYS 76 Ile 17 Glu 78 Asp 79 Ser 80 Asp 81 Thr 82 Tyr 83 Ile 84 CYS 85 Ghl 86 Val 87 Glu 88 Asp 89 Gln 90 LYs 91 Glll 92 Glu 93 Val 94 Gln 95 LeU

96 LCU

97 Val 98 Phe 99 GUY

‘fL

aL

*s, aL

OL *aL EL

BB:: YL UL aL

CuL

aL

aL

bL cue CL

VL

CL

+YL

*YL

*YL

*aL +YL *YL EL aL

UL aL

‘72 8L CL EL 8L CL YL YL 'YD +aL CL CL *hL 4

YL

*YL

eL +YL

CL 8L

8L

CL

(IL 6L

aL

aL aL

CL 8L 8L YI.

QL QL aL aL QL aL

aL 8L 6L 61.

8L YL

YL

aL YL LI.

8L w aL

aL

CL 8L YL

YL

EL

YL

YL

YL

CD ffL

YL

YL

CL

8L

8L

CL

YL

YL

PI,

8, 8L

8L

aL aL

aL

aL

OL fL BL 8L YL

QL QI.

QL aL

QL SL aD YL YL (1.

YL YL YL

CL QD CL

8L aL

QL QL t

8L YL

tL YL

EL

BL

YL

70

aL

eL 8L YL

YL

YL

YL

tL YL

CL EL YL

8L+

aL

cuL* aL

aL

6L* CL BL 8~

YL

QL

aL

UL

QL

aL

bL

aD

;*

CL

fL*

CL*

BL* CL*

CID*

CL*

eL

aL

aL

aL

CL*

bL CL

;I”c.

tL

YL

YL

CD*

bL*

EL

EL

8t CL

YL

CL+

CL

CL’

CL

8~ 8~

Z. Sz&ely et aLlJournal of Molecular Structure (Theochem) 367 (1996) I.59-I86 167

Table 2 Table 2

Continued Continued

No. AA lcd4 3cd4

x-ray MM MM x-ray No.

151

152

I53

154

155

156

157

158

159

160

161

162

163

164

165 166

167

168

169

170

171

172

lcd4

AA x-ray

Leu

Gltl 1;;

ASP aL Set *YL

Gly *CL

Tbr *YL

Trp EL

lbr *YL

GYS CL

lbr BL

Val CL

LCU *CL

Gln BL

Asn +YL Gln l YD

LYS CL

LYS BL

Vat CL

Glu +YL

Phe CL

LYS YL

Ile *en

loo LeU

101 Thr

102 Ala

103 Asn

104 Ser

105 Asp 106 Tllr

107 His

108 LXX

109 LeU

110 Gln

111 Gly 112 Gln

113 Ser

114 LCU

115 Thr

116 LCU

117 ‘Ihr

118 LCU

119 Glu

120 Ser

121 Pro

122 Pro

123 GUY 124 Ser

125 Ser

126 Pro

127 Ser

128 Vat

129 Gln

130 CYS 131 Arg 132 Ser

133 Pro

134 Arg 135 GUY 136 LYS 137 Asn

138 Ile

139 Gln

140 Gly 141 GUY 142 LYS 143 Tnr

144 LCU

145 Ser

146 Val

147 Ser

148 Gltl

149 LeU

150 Glu

YL

CL

‘CL

QL

+BL

*YL

*YL

‘-lx

CL

*CD

‘CD

*70

CL

CL

CL

*SL

EL

-1

CL

eL

BL

FL

(YL

*bL

VL

*bL

EL

eL

CL

BL

CL

BL

CL

*YL

*CD

‘CD

*aD

‘CL

EL

BL

BL

*6D

ffL

‘EL

CL

*b

YL

+YL

*aD

CL

BL

(I. fL

BL YL

BL CL

aL 61

BL YL

tL 7D

YL BL

YL =D

CL 7L

(YD CL

8 4

7n YO

CL fL

YL fL

YL CL

YL BL

CL BL

BL eL

YL YL

Q. (L

BL BL

fL fl.

aL QL

aL YL

EL aD

YL CL

CL CL

CL CL

YL BL

YL 6L

YL EL

YL (1

CL CL

YL aL

CD BD

YD YD

(L ZL

BL CL

I%. BL

BL CL

81. I%.

CL BL

6L aL

BL BL

BL BL

YL BL

YL 81

YL aL

OLD BL

YL EL

YL CL

YL

tL

YL*

aL

sL*

YD*

BL*

&D’

eL

CL*

9’

aD*

EL

CL

CL

CL*

Bz*

CL

t1

BL

EL

a1

YL’

CD’

BL*

CL

CL

CL

BL

CL

IgL

0

aL+

h’

YD*

CL*

YL*

EL

BL

BL

CL*

aL

BL’

CL

&*

YL

aL*

BL*

CL

BL

MM

BL

BL

aL

aL

YL

tL

CL

YL

YL

YL

YL

Q.

CL

CL

7D

EL

BL

2

BL

BL

CD

3cd4

MM

aL

aL

YL

CL

CD

YL

YL

Yl.

YL

CL

CL

(1.

BL

YD

2

YL

CL

BL

eL

BL

BL

x-ray

aL*

aL+

aL

tL*

BL*

8L’

fL

I%.*

CL

BL

CL

YL+

BL

CD*

aL+

CL

BL

CL

BL*

CL

Y

c

dielectric constant and (3) with a 15 A cut-off of the nonbonded interactions. During structure optimisa- tions (4) a conjugate gradient algorithm was used with a 0.001 (kcal mol-’ A-‘) derivative convergence criteria. At (5) 310K equilibration temperature, the following options were used: 1 fs time step, 110 ps total time, recompute neighbour list of residues every 20 steps and history output every 200 steps for dynamic simulations.

The Delphi program [29] was utilized for the electrostatic calculations using a SGI ONYX workstation. In the intramolecular space E = 4, in the extramolecu- lar space E = 80 were used for dielectric constants.

The MD resulted in Cartesian coordinates which were converted to backbone torsional angles (+,$) according to IUPAC-IUB convention. These torsional angle pairs were then assigned to one of the 9 ‘conformational centres’ as described elsewhere [31,32]. The codes and the locations of the 9 ‘conformational centres’ are summarised in Table 1. The Amino Acid Conformation Assignment of Proteins (ACAP) software [33a,b,c] has been used to analyse the conformational change of the 35-60 backbone segment of the

168 2. S..&ely et al.lJournal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 3 X-ray discrepancies of CD4 that may or may not be converted to the same conformation domains after MM optimization. The interconnections between ridges are marked by /

No. AA X-ray

2.3 A

MM

2.3 A 2.2 d;

Ridges

3 Val 4 Val 5 Lell

6 GUY 12 Val 17 Thr 18 Ala 20 Gin 22 LYs 24 Be 25 Gln 31 Ser 32 Asn 33 Ghl 39 Asn 43 Phe 45 Thr 46 LYs 47 GUY 49 Ser 51 Leu 54 Arg 67 Phe 69 LeU

70 Ile 71 Ile 72 LYs 73 Asn 74 LeU

79 Ser 87 Glu 88 ASP 91 Glu 94 Gln 96 Leu 102 Ala 104 Ser 105 AsP 106 Thr 107 His 109 J&l

110 Gln 111 GUY 115 Thr 117 Thr 123 GUY 124 Ser 125 Ser 133 Pro 134 Arg

BL 8, Yr

EL

EL

YL

7‘

8,

EL

YL

7‘

ii:

6, Yr 8,

7‘ EL

ED

EL

6, a‘

7‘ 7‘

7‘

7‘ a‘ 7‘ YL 7‘ 70

;:

7‘ 7‘ EL BL 7‘ 7‘ Yr ED en 70 8, EL 6, PL 6, 7‘ ev

2 Yr CL

8,

YL

YL EL

EL

a‘

EL

Yr 6, 6, Yr EL

A BL ED

EL

6, a‘

2 YL

7‘ a‘

Yr EL EL ED (YL CL CL YL

Z:. EL 7‘ 7‘ a, El, 70 7‘ BL QL EL 7‘ 7‘ ED

;: EL

EL

A Yr 6‘

a, 8, EL

EL

a‘

a‘

a0

8,

7‘ EL 7‘ EL BL a‘ a‘ Yr 7‘ 7‘ 7‘ 6‘ a0 EL EL 70 ff‘ Yr 7‘ YL EL 7‘ YD 8, a, EL 6, 70 8, CL 7‘ ffD EL (YL PL

A A A A B B B/C B/C WC WC WC C/C’ C/C’ C/C’ C’ C’ c c c”/D c”/D c”P D E E E E E E/F E/F E/P PIG PIG G A A A AIB A/B 4/B A/B 4/B 4/B AA B B B/C WC WC C/C’ C/C’

Z. Sdely et al.lJournal of Molecular Structure (Theochem) 367 (19%) 159-186 169

Table 3

Continued

No. AA

135 GUY 136 LYs 137 Asn 141 GUY 143 Thr 145 Ser 147 Ser 148 Gln 151 L.eu 152 Gln 154 Ser 155 GUY 156 Thr 158 Tllr

162 LfX

164 Asn 165 Gln 169 Glu

172 lle

Ridges

C/C’ C/C’ C/C’

C’/E E E

E/F E/F E/F E/F

E/F F F F F

FIG F/G G G

Dl domain during 110 ps change of the MD analysis using 5 ps sampling.

The ab initio molecular orbitals (MO) were computed on a HP 755 computer using the GAUSSIAN 92

program [34]. Three standard basis sets [35-381 were used: 3-21G, 6-31G and 6-31 + G’*.

4. Results and discussion

4.1. A conformational comparison of CD4 crystal structures

The DlD2 superdomain of CD4 was ftudied previously by X-day crystallography to 2.3 A @dblcd4) [14] and 2.2 A (pdb3cd4) [16] resolution. Both structures, taken from the Brookhaven Protein Data Bank, were subjected to a backbone conformation assignment analysis. All conformational building units of the main chains of the proteins were categorised into one of the 9 basic conformational types (Table 2). The subconformers are reported in terms of sub- scripted Greek letters (i.e. aL, cr,, P,, etc...), as suggested earlier [31-331. Those sequential regions, where a conformational mismatch was observed

between the two crystal structures, are marked by asterisks (*). (if further refinement of the CD4 X-ray structure is deemed necessary, then the amino acid residues marked by asterisks (‘) would be the most likely sites where improvement might be achieved most easily.) The reported crystal structures were sequentially submitted to MM geometry optimisation. Due to the applied force field, some of the discrepancies in the X-ray assignments disappeared. The sites, where the conformational assignments are in agreement with the MM geometries, are presented in bold in Table 2. All those amino acid residues which are marked by asterisks ( l ) in Table 2 are summarised in Table 3.

4.2. Internal salt bridge formation in the Asnj2Asp mutant

The expression of the Asn’*Asp mutant [39] of CD4 led to some remarkable experimental results, providing a simple explanation of the observed change in the binding affinity. It appeared that the exchange of Asn to Asp at position 52 induces a direct re-orientation of the Lys& side-chain, namely that the intermolecular electrostatic attraction between gp120

and CD4 will be replaced, at least initially, by the formation of an intramolecular (-NH3+***-OOC-) salt-bridge. Subsequently, a hydrogen bonded complex (-H2N:...HOOC-) within the CD4 molecule may be formed without the dramatic modification of the backbone conformation. More specifically, this model suggests that the original secondary structure of the {35,60} sequential region of CD4 is preserved even in the Asn”Asp mutant. Such a hypothesis has been corroborated during a MD simulation, since no major conformational alteration was observed for the Asn5*Asp mutants respect to wild-type CD4 (WT)

PI. Table 4 summarises the observed conformational

shifts of an interesting sequential region of the WT CD4 during MD, with a 5 ps sampling frequency. These data reveal that most of the conformational changes occur in the segment (46-54) that intercon- nects ridge C’ and ridge D (c.f. Fig. 2). Based on the MD simulations a direct comparison was achieved between the Asn’*Asp and the wide-type CD4 in

this most interesting {35-60) sequential region (Table 4 and Table 5). During MD simulation the same sequential subunit may show a different flexibility character, depending on the amino acid at position 52 (Table 6). The preceding Ser-Lys-Leu (48,49,50) unit have a more rigid (R) backbone conformation in the Asn5*Asp mutant, than computed in the WT. The inverse gamma turn (yJ and the extended like (@, eL) subconformation of Ser-Lys-Leu is preserved during the entire 110 ps. On the other hand, the more flexible (F) nature of the same tripeptide in the WT CD4 is reflected by the observed conformational fluctuation. Alternative backbone conformers (at 49 yL -+ 0, yL (r& were observed for the same amino acid using similar MD conditions.

The earlier MD results [40] for the segment {41- 60} of the Lys@‘Ala mutant shed some light on the conformational importance of Lys&. This is the amino acid which is involved in the intramolecular ‘salt bridge’ formation within the Asn5*Asp mutant.

The intramolecular salt bridge formation in

2. S.Gely et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186 171

Table 5 Conformational assignments to the molecular dynamics (MD) results for the Asn”Asp mutant of CD4 in the region of Lys35-Ser60

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110

Asn5’Asp mutant shows a marked change in charge distribution with respect to the WT.. Fig. 7(a) shows that the WT had an appreciable amount of positive charge due to the free Lys6 side chain. In contrast, the excess positive charge in the vicinity of Lys# is neutralized, at least partially, in the Asn52Asp mutant due to the As~~~..*Lys~ intramolecular salt-bridge formation as shown in Fig. 7(b).

4.3. Electrostatic attraction as the basis of pre- orientation

At the pH level of the blood @H 7.4) all the amino and guanidine groups of the side chains in lysines and arginines, respectively, are fully protonated. Since the CD4 molecule is positively charged, therefore one may assume that the surface of gp120, at least at the binding region, may carry negative charges. The CD4

Table 6 Rigid (R) and flexible (F) backbone conformations in the 45-55 segment of the wild-type (WT) CD4 receptor and its Asn”Asp mutant

Ridge cc

Interconnection Ridge D*

xs2 45 46’ 47 48 49 50 51 52’ 53 54 55 Thr LYS Gly Pro Ser LYS LeU X JQ+P kg Ala

Asn R R R F F F R R F F R WT VI EL PL LYL,OLL &YL,% Em-f, (YL aL VO&, &>ff, EL

ASP VI EL 6 dL>V, YI PL E, ffL@D,YD UL “DVO ffLA EL mutant R R F R R R F R F F R

’ ‘salt bridge’ or hydrogen bonded complex.

172 Z. S.z&ly et aLIJourna1 of Molecular Structure (Theochem) 367 (1996) 159-186

Fig. 7. Electrostatic 3D-grid: (A) Wild type Dl and D2 domains of the CD4; (ES) Asn”Asp mutant Dl and D2 domains of the CD4. For the ease of visualization a solvent accessible surface was created. This ConoIly surface was colored as red, blue and white for positive, negative and neutral.

Z. Sz&ely et nl.lJournal of Molecular Structure (Theochem) 367 (1996) 159-186 173

CD4

Phase A I

Lys”-NH,+

Lys4”-NH?+ -OOC-Glu””

A~u~~-C f NH,

D -OOC-Asp3”* \

NH,

‘salt-bridge’

gp120 -_) CD4

Phase 82

Ly&N: . . . . . . \

HOOC_Asp457

H

H Lys4”_N:l . .._... HOOC_Glu37”

H

Arg’“-C f

NH2 ...‘. 0,

\ - ,C-AspJ6s

NH, ‘.‘.. 0

Brensyed (H-bonded) complex and ‘salt-bridge’

:

gp12O

Scheme 3. Transition from “salt bridge” to H-bonded complexes.

and the gp120 do bind to one another throughout the contact involving the above amino acid residues, which is further corroborated by the fact that the gp120 itself cannot bind to the CD4 when the positively charged Lys or Arg are replaced by alanine. This is clearly observed when at strategic positions LYS~~, Lys3’, LYS~~ and Arg59 are changed to alanines

WI. It seemed reasonable therefore, that the positively

charged LYS~~, Lys6 and Arg59 of the CD4 would be attracted by the negatively charged charged ASPIRE, GAUGE’ and Asp”7 of the gp120, as illustrated schematically in Fig. 5.

The pre-orientation is expected to occur before the gp120 can actually bind to the CD4. The notion of pre-orientation is based on the idea that in molecular recognition atoms do not see atoms, but the electrostatic field of one of the molecules experiences the electrostatic fields the other molecule. The character- istic positively charged surface on the Dl domain of CD4 is illustrated in Fig. 7. The result of the pre- orientation is that the two molecules orient themselves relative to each other so that opposite charges might meet (phase Al).

4.4. Electronic attachment

In phase B2 there is a short range electrostatic interaction which may result in a change from salt bridge formation (c.f. Scheme 3) to a Bransted (H-bonded) complex formation. This is achieved for the lysine

residues as shown schematically in Scheme 3. Earlier ab inito MO study has shown [19] that a

neutral Bronsted complex between an amine and a carboxylic acid is more than 10 kcal mol-’ more stable than the zwitter-ionic salt bridge (between a protonated amine and a carboxylate ion), at least in the gas phase. It remains to be seen if solvation will reverse the stability and the salt bridge will become more stable. More sophisticated ab initio study is needed in order to answer that question. However, irrespective of what type of bonding may exist between the components, there must be a geometrical complementary between the 3 a-carbons of the positively charged amino acids of CD4 and the 3 o-carbons of the negatively charged amino acids of gp120.

The mirror image topological arrangement is expected in the gp120 for the amino acid residues with the carboxylate side chains, so that Asp457 may

Lys3’ ;

f ; 17.5 A

J T ‘?\

Arg5” * 8.k : ? -AspJhX

f \

13.48,

i i /’

k Ly? : &‘C,Y ?

Scheme 4. Spatial arrangement of the a carbons associated posi-

tively charged side chains of CD4 (left) and of negatively charged

side chains of gp 120 (right).

174 2. S&ely et al.lJoumal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 7 Complexation equilibrium constant (l/KJ for gp120 and CD4 mutants

Mutant

Phe 43Ala Phe “Ile

Amino acid Side chain (1/Kd)10-9

Ala -CH3 0.002 Ile -CH--CH2-_CH3 0.002

I

CH3

Phe43Lcu 30

J_eu /m3

-CHdH 0.400

a P ’ CH3

WT Phe -CH 1.111

be matched up with LYS~~ and GAUGE’ may perhaps be paired up with LYS~~ etc. as illustrated schematically in Scheme 4.

4.5. Phenylalanine (Phe43) as a ‘docking lock’

One of the aims of this paper is to underline the crucial role of Phe43 as a ‘docking-lock’, which is the final step of the docking (B3) that takes place after the long-range (Al) and short-range (BZ) electrostatic attraction.

The benzyl-group (-CH2-Ph), which is the apolar side chain of the phenylalanine residue, has been altered previously [7] by point mutation in CD4. Three different but all apolar side chains: -CH3 (Ala), -CH(CH3)-CH2-CH3 (Ile) and -CH*-CH2- CH(CH& (Leu) were introduced [12]. Such muta- tions reduced the complexation between gp120 and CD4 as defined by the reciprocal value of the disso-

Table 8 Conformational assignments to the molecular dynamics (MD) results for the Pheh3Ala mutant of the CD4 in the region of Lys3’-Serw

0 5 10 15 20 25 30 3.5 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110

2. Szikely et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186 17s

Scheme 5. Eight patterns of conformation hopping which were observed during the MD simulations of the WT and the mutants of CD4 (see

Tables 6 and I1 for details).

ciation equilibrium constant, l/Kd;

1 /KA -

gp120 + CD4 Z gpl20**CD4(complex)

The actual values are presented in Table 7. It appears that the Leu side-chain with its, tertiary (3’) C-H bond of the 0 carbon, is still effective, to &me begree, in the complexation.

Kd These biological results suggest that the dynamics

Table 9

Conformational assignments to the molecular dynamics (MD) results for the Phe4’11e mutant of CD4 in the region of Lys”-Serho

0 5 10 15 20 25 30 35 40 45 SO 55 60 65 70 75 80 85 90 95 100 105 110

176 2. S&ely et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 10 Conformational assignments to the molecular dynamics results for the Phe43Leu mutant of CD4 in the region of Lys3s-Serh”

nature of these three mutants should reveal interesting aspects of the molecules and particular attention

should be given again to the (3.5-60) segment of the CD4.

Tables 8-10 report the MD results obtained on the Phe43Ala, Phe4311e and Phe43Leu mutants respec-

tively. Table 11 summarises the structural concIusion of these MD results presented in Tables 8-10. Con-

sidering the amino acid residues from 3.5 to 50, the eight patterns of ‘conformational hopping’ (shown in Scheme 5) have been observed involving two or three nearest-neighbour conformational centres.

Table 11

Rigid (R) and flexible (F) backbone conformations in the {35-50) segment of wild-type (WT) CD4 receptor and its Phe43Ala, Phe4111e and

Phe43Leu mutants

X”

Ridge C’

35 36

Lys Ile

37 38 39

Leu Gly Asn

40

Gin

41 42

Gly Ser

Phe43

WT

Ala@

mutant

Ile43

mutant LeU”

mutant

Ridge c”

43 44 45 46 47

X Leu Thr Lys GUY

49

Ser

Z. Szkkely et alJournal of Molecular Structure (Theochem) 367 (1996) 159-186 177

H\ ,9 H-C,-CrC"

It b2

p-H2

H,-C <

02

2

Y p,- H2

H >G-C$

H 02

4

6

Y3 H,- N\! P

,C= N, H,- N

I’ H4

7

H4

8

Scheme 6. Conjugated base and conjugated acid forms of the

selected model compounds.

With regards to residue 43 only the Phe43Ala backbone conformation turned out to be rigid (R), while

the Phe4311e and the Phe43Leu mutants, together with the wild-type (i.e. Phe43Phe) demonstrated flexible

backbone conformations oscillating between the yL and its neighbouring eL conformational centres. This would suggest that the flexible side chains, -CH*-Ph,

-CH2-CH(CH& and -CH(CHs)-CH2-CH3 in Phe, Leu and Ile respectively, induced some backbone flexibility. Clearly, a similar transmission of flexibility

from side-chain to backbone was not possible in the case of a methyl group (Ala). Perhaps, this is related to the fact that the -CH3 side-chain has only one unique

conformation. Consequently, the flexibility of the backbone observed in Table 11 appears to have noth- ing to do with the aromatic ‘docking-lock’ mechanism, but it is related to the side-chain-backbone

flexibility coupling, or more precisely the coupling

between the {A$} and {x1,x2,...) subspaces.

4.6. Ab initio MO results

In the present ab initio study, which is exploratory in its nature, H-COO- and CH3-COO- were used to

mimic glutamate and aspartate. Similarly the side- chains of the protonated lysine and arginine were mimicked by +H2N-CH3 and ‘H*N-C(NH& It

might be more appropriate to use N-methyl guanidinium ion as a model, including the terminal carbon of

the amino acid side-chain, but the geometry optimisa-

tion of the guanidinium ion was easier due to its sym- metry. The structure of the four unprotonated and four protonated species (numbered from 1 to 8) are shown in Scheme 6. Their optimised geometrical parameters and computed energy values are summarised in the Table 12Table 13Table 14.

The first question to be answered was the relative

gas phase basicity of amine and guanidine versus the carboxylate ion. On the basis of the data presented in Tables 12-14, energy level diagrams may be con- structed in which the energy differences represent

proton affinities or proton affinity differences. Such an energy level diagram is shown in Fig. 8. It may be

ascertained from this figure that any one of the negatively charged carboxylate ions has a grater proton affinity, therefore more basic, than either one of the neutral nitrogen containing bases. In all cases, the difference is in excess of 100 kcal mol-‘.

When the two nitrogen containing bases are com-

pared, guanidine turned out to be more basic than methyl amine, implying that protonated guanidine is less acidic than protonated methyl amine, at least in

the gas phase. The difference is in excess of 25 kcal - mol-’ (i.e. 253.8 - 227.9 = 25.9).

Such a difference may be one of the reasons why proton transfers occurs in the case of a protonated

amine, and a hydrogen bonded complex is formed between two neutral components CH3-NH; + -OOC-CH3 - CH3-NH2 + HOOC-CH3 as it also

became evident in the earlier publications [18-231. However, no proton jump was observed in the case of guanidine and the carboxylate ion leading to zwit-

terionic ‘salt-bridge’ type complex formation. Another possible difference may be due to the fact

that in the case of -COO- and +H3N- there is a mis-

178 Z. S.&kely et al.IJournal of Molecular Structure (Theochem) 367 (19%) 159-186

Table 12

Optimized geometrical parameters and total energies of selected carboxylic acids R-COOH (2,4) and their anions, R-COO- (1,3)

Geometrical parameters (6-31 + G”)

Dimension 1 2 3 4

HCI HI& C&2

c201

c202

%H2

HClG

H&z01

H&202

ClC2Ol

GC202

OK202

H201C2

- 1.119217 -

1.234690

1.234690

- 114.774

114.774 -

- 180.000 -

- 1.084135 -

1.321401

1.183687

0.949472

-

110.719

124.511 -

-

124.7703

109.579

- 180.000

0.000

1.08 1.08 - -

1.546361 1.500538

1.238080 1.331190 1.238052 1.189033 - 0.948369

110 109.5 - -

- 115.608

115.642

128.7308 -

181.464 -

-

112.117

125.685

122.1981

108.891

180.002 -

- 0.002

Total energies (Hartree)

E(6-31G) - 188.0951541 - 188.6654882 - 227.1225717 - 227.7011158 E(6-31 + G”) - 188.2097438 - 188.7776531 - 227.2543528 - 227.8294868

Starting geometry 1

Starting geometry 4

Starting geometry 2 Starting geometry 3

Optimized geometry: from $2 and 3.

+

H\ ,qu. H r-4 ,H5 Starting geometry 5

H-,G--G:? %,Cs N3

H \q..H.+ ‘s

H4 Optimi.e;n~~metry:

Scheme 7. Starting and optimized geometries of complex models.

Z. Wkely et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186 179

-283.2, I !

OH --323.2 a

I I

( ) a s

& $ b) >

Formate-methyl amine proton affinity Acetate-methyl amine proton affinity ~3

IT [r oz w w

= -392.2- H_+; H@ NH2 HN+

: H&-C!; t?

NH;! - Y

HN=( -431.2 w

;: NH2 a

---------------- /- -392.4-

T

0

0 1 I- 253.8

_ j_ _ H-$+H2N+;z

--431.8

-393.0- Jl NH2 H-C + HN<

‘OH NH2 -393.2

( 1 C

--400 -4oo- /P

H&-C, OH +“H:

t HN - -432.2

(d) Formate-guanidine proton affinity Acetate-guanidine proton affinity

Fig. 8. Energy level diagrams for protonation. The total energies are given in hartrees. Energy differences corresponding to PA and APA are given in kcal mol-‘z units.

match of the ligands: 3 positively charged H meet with two negatively charged 0. In contrast to that there is a perfect match when -COO- meets with +H2N-C(NH2)2, as 2 positively charged H meet with 2 negatively charged 0. The situation is depicted for formate and acetate ions in Fig. 9.

It should be emphasised that in the present paper this difference in the complexation mechanism of the protonated amine and the protonated guanidinium was re-examined. Three initial geometries were studied for the acetate-protonated methylamine complexation

(denoted as ‘starting geometry’ 1,2 and 3) and each one converged to the same optimised geometry corresponding to the hydrogen bonded neutral complex.

Similarly, two geometries (denoted as ‘starting geometry’ 4 and 5) corresponding to the co-planar and perpendicular arrangements, have been used for the acetate-protonated guanidine complex, and both of these converged to the same optimised geometry corresponding to the ‘salt-bridge’ zwitterionic complex. All of these are depicted in the following scheme (Scheme 7).

180 Z. Szikely et al.IJournal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 13

Optimized geometrical parameters and total energies of methyl

amine (5) and its protonated form (6)

Geometrical parameters 6-31 + G”

Dimension 5

HlN 0.999894 HzN 0.999894 H3N - NC 1.45319s CH 1.085

H,NC 111.400

H2NC 111.400

H3NC - NCH 111

HWZ 107.4752


6

1.010073

1.010073

1.010073

1.505884

1.078557

111.477

111.477

111.477

108.193

107.393

E(6-31G) - 95.168939 - 95.5388005 E(6-31 + G”) - 95.2262788 - 95.5895351

Table 14

Optimized geometrical parameters and total energies of guanidine

(7) and its protonated form (8)

Geometrical parameters (6-31 + G”)

Dimension 7

HINI 0.990778

H2Nz 0.989472

H~NI 0.990626

H4Nz 0.991453

HsN3 1 BOO774

H&3 -

NIC 1.370947

N2C 1.363032

NJC 1.268555

HlNiC 122.291

H2NzC 123.204

H3NlC 119.791

H4NzC 117.330

HsN~C 112.327

HBN~C

NKNz 114.159

N&N, 126.180 N&N2 119.661

HsNxCNI 0.000


8

0.995273

0.995273

0.995273

0.995273

0.995273

0.995273

1.322145

1.322145

1.322145

121.459

121.459

121.459

121.459

121.459

121.459

120.0

120.0 120.0

0.000

E(6-31G) - 204.0331102 - 204.1456561 E(6-31 + G”) - 204.4507174 - 204.5501665

Reactants(R) Product(P)

,? H-q.0 + H&CH,

/o-l+ \‘<‘H (IA)

0 __t H-c*O_____~/ kH3

0 CH3-e + H.$CH,

? 7 0

H-G4i3 + H-N\ O----H-N,

__t H-C% (IIA)

Y3

e,;C-NH, @J- NH2

H-N ~0:-_+-pJ

il A

7 Y 0

CH,-C% + H-TX

(s:C-NH, <?---H-N,

CH,-CqQ W-NH, (IIB) qO H-lrj/ ‘O___H_N’

k I:

Fig. 9. Reactions for hydrogen bonded and ‘salt bridge’ type com-

plexations.

Table 15

Optimized geometrical parameters using starting geometry 1

Dimension 3-21G 6-31G 6-31 + G”

C&z

GN

C IH 6%)

GOI

c202

NC3

OIHI

02H2

HIN

H2N

H3N

C3H (w)

WC2

WZOl

ClC202

01c202

C3NC2

HW3

HW3

HW3

1.504216

3.391551

1.08

1.330522

1.214079

1.492328

1.014092

3.446611

1.668104

1.007872

1.006899

1.08

109

111.647

124.634

123.7183

88.4655

105.4017

111.6016

112.773

180.006

177.960

39.517

219.056

1.492741

3.441397

1.08

1.334130

1.220647

1.467996

0.986775

3.141393

1.763280

0.998851

0.999899

1.08

110

112.994

124.454

122.5514

95.3471

105.3404

113.0355

114.1043

179.914 179.669

63.882

213.839

1.503101

3.434800

1.08

1.316619

1.196204

1.458375

0.967475

2.641291

1.900247

1.002640

1.000905

1.08

109.5

112.522

124.232

123.2466

122.6596

117.33

111.1244

111.0614

179.990

180.519

96.231

195.744

E(Hartree) - 321.2461075 - 322.8934246 - 323.0702849

Z. Szikely et alSJourna1 of Molecular Structure (Theochem) 367 (1996) 159-186 181

Table 16


Dimension 3-21G 6-31G 6-31 + G*’

Table 17


Dimension 3-21G 6-31G 6-31 + G’*

1.492319 3.374795 1.08 1.334716 1.221170 1.464591

0.985552 2.587222 1.775997 1.001310 0.998731 1.08

1.503153 3.440267 1.08

1.316621 1.196154 1.458307 0.967555 2.665000 1.898784 1.002553 1.000866

1.08

GIG! 1 SO2464 C2N 3.202238

C IH (avg) 1.08 CZOI 1.332444

c20: 1.215750 NC3 1.483592

GIH, 1.009960 O?H? 2.237264 HIN I .694730 HZN 1.009694 HJN 1.006448

CAH (avg) 1.08

HC,C2 109 C,CZOl 111.972 C,CZOL 124.841 O,CZOL 123.1872 C~NCZ 123.5749 H,NC? 115.709 H~NCJ 112.8529 HjNC? 112.5386

O,C~C,O~ 179.961 N&O,C, 180.493 C,NCZOI 96.396

H-INCKZ 192.322

E(Hartree) - 321.2460592 - 322.8935084 - 323.0702838

110

113.049 124.464 122.4866 119.4661 112.3018 113.9543 113.9463

109 112.520 124.235 123.2457 122.068

116.8515 111.1403 111.1044

180.026 180.196 99.655

203.747

180.037 180.365 96.160

196.690

Cl& 1.502454 1.492303 1.503154

W 3.204507 3.379129 3.440398

C IH (avg) 1.08 1.08 1.08

c201 1.332543 1.334835 1.316611

c202 1.215692 1.221099 1.196145

NC3 1.483500 1.464618 1.458391

O,H, 1.009873 0.985370 0.967589

02Hz 2.242701 2.593218 2.668155

HIN 1.695750 1.779366 1.898271

H2N 1.009576 1.001276 1.002536

Hd 1.006375 0.998739 1.000859

Cd (ad 1.08 1.08 1 .08

HCiC2 109 110 109.5

ClGOl 111.971 113.045 112.520 c*c202 124.854 124.473 124.234

01c202 123.1749 122.4822 123.2458 C3NC2 123.2888 119.4493 121.6513

H,NCj 115.5752 112.0285 116.6426

HzNCj 112.8909 113.9952 111.1421

H,NCx 117.5804 113.934 I 11.0936

o,c>c,o2 180.015 180.023 180.007 NCzOIC, 179.410 179.434 179.260 C,NC201 102.633 114.524 107.432

H3NC& 188.265 195.44 193.516

E(Hartree) - 321.246058 - 322.89351 - 323.0702827

The geometrical parameters and the associated energy value obtained at the HF/3-21G, HF/6-31G

and HF/6-31 + G*’ levels of theory are summarised in Tables 15-19 corresponding to starting geometries

1, 2, 3, 4 and 5 respectively. Clearly starting geome-

tries 1, 2 and 3 (presented in Tables 15-17 respectively) converged to the same structure which is the hydrogen bonded (Brensted) complex. Similarly, starting geometries 4 and 5 (presented in Tables 18

and 19 respectively) converged to the same structure, the zwitterionic ‘salt-bridge’ complex. The energies

of the various complex formations, as depicted in Fig. 9, are summarised in Table 20.

At last, but not least, the pre-orientation process was questioned. The different cross-sections of the potential energy hypersurfaces (PEHS) clearly

show in Figs 10 and 11 that indeed there is a pre- orientation for the charged moieties, since the system would most likely choose the reaction coordinate, (selecting between most favourable cross-sections,

that would correspond to something like steepest descent).

4.7. Internal interaction

Finally, a few words have to be said about the ‘locking-in’, via aromatic ring orientation (B3). It should be emphasized that it is not known precisely,

at this time, whether two or three aromatic rings are involved as illustrated in Fig. 12.

In either case ‘aromatic stacking’ may be the mechanism for such a ‘locking-in’ mechanism. How-

ever, if the three aromatic ring mechanism is opera- tive, then an alternative mechanism must also be considered. This alternative mechanism would involve the phenolic OH of Tyr435. Benzene rings can form hydrogen bonded (Brpnsted) complexes. This is illustrated recently [36] for the case of the benzene ring and water. If C6H6*-.HOH does form a hydrogen bonded complex, then the stabilization for

182 2. SztMy et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186

Table 18 Optimized geometrical parameters using starting geometry 4

Dimension 3-21G 6-31G 6-31 + G”

Table 19 Optimized geometrical parameters using starting geometry 5

Dimension 3-21G 6-31G 6-31 + G”

ClC2 1.520571 1.506326 1.516938

cZc3 3.824488 3.899684 3.886832

C IH Wg) 1.08 1.08 1.08

c201 1.259343 1.266072 1.244124

w2 1.262984 1.269139 1.246330

081 1.532870 1.614968 1.622132

082 1.515470 1.601485 1.611058

N1C3 1.315201 1.318691 1.312437

N2C3 1.314051 1.317930 1.311809

N3C3 1.353528 1.351668 1.346563

NJ-II 1.065553 1.041271 1.041880

NcH3 0.996659 0.991364 0.992504

N2H2 1.070947 1.043886 1.043954

N2I-b 0.996822 0.991458 0.992562

N3Hs 0.995346 0.990280 0.991679

N3& 0.995342 0.990277 0.991681

HCKz 109.5 110 109.5

ClCZOl 118.087 118.616 118.147

w2oz 116.809 117.597 116.922

OlC202 125.1022 123.7848 124.9287

HtNK3 120.8919 121.0461 120.5133

H3NK3 120.4127 120.8752 120.116

HzN2C3 120.9786 121.043 120.5207

Hfi2C3 120.3214 120.8435 120.0667

&N&3 121.1761 121.2844 121.0311

H83C3 121.1491 121.2662 121.0159

N3c3N1 119.6436 119.6361 119.7795

NlW2 120.5696 120.6354 120.3674

OlC2W2

c3c201c1

N1C3C201

N2C3CZ02

N3C3NKz

HNCN

180.507 180.509 180.435 01c2c102 180.108 179.634 179.675 179.643 ~3~2~1~1 179.945

0.030 0.092 0.011 N1C3C201 - ,037 0.010 0.003 0.005 N2C3C202 - ,082

180.159 180.119 180.096 N3C3NK2 180.010 180.0 180.0 180.0 HNCN 180

ClC2 1.520581 1.506346 1.516906

c2c3 3.824503 3.899243 3.887006

CIH (w) 1.08 1.08 1.08

c20, 1.263066 1.269168 1.246392

c202 1.259259 1.266010 1.244111

OIHI 1.515080 1.601207 1.611177

02H2 1.533152 1.614565 1.622356

N1C3 1.314019 1.317908 1.311814

N2C3 1.315229 1.318680 1.312428

N3C3 1.353540 1.351673 1.346561

NIHI 1.071059 1.043918 1.043968

NlH3 1.065431 1.041283 1.041878

NzH2 0.996823 0.991456 0.992564

N2b 0.996652 0.991367 0.992505

N3h 0.995342 0.990279 0.991680

N3H6 0.995345 0.990280 0.991679

WC2 109.5 109.5 109.5

ClC201 116.779 117.577 116.893

C&202 118.120 118.634 118.176

w202 125.1005 123.7892 124.9313

HINIC~ 120.976 121.052 120.520

H3NtC3 120.320 121.056 120.506

H2NzC3 120.895 120.832 120.066

bNzC3 120.412 120.866 120.116

W’J3C3 121.149 121.268 121.015

hN3C3 121.176 121.285 121.032

N3GN1 119.7952 119.7193 119.8533

NiC3N2 120.5643 120.6409 120.3668

180.110 180.091

179.952 179.946 0.052 - .076 0.000 - ,090

180.015 180.019

180 180

E(Hartree) - 429.5598529 - 431.7748413 - 431.9913664 E(Hartree) - 429.5598534 - 431.7748413 - 431.9913644

C6H6**.HOAr must be even more pronounced since the phenolic OH proton is more acidic than the proton in HrO. Future ab initio computation will be in the position to decide if this mixed Lewis/Brpmsted complex, as outlined above and as shown in Fig. 12, is more stable than the aromatic stacking (pure Lewis complex) involving three nearly parallel aromatic rings. However, the explicit involvement of Tyr435 can only be assessed if and when that particular tyr- osine residue is replaced by a non-phenolic apolar residue such as alanine (i.e., Tyr435Ala) in the gp120 by point mutation.

5. Conclusions

The two X-ray structures of highest resolution, though they showed some improvment in terms of the fewer discrepancies observed, nevertheless, the two structures were not widely different from that of the previous pair of structures, as could be judged from the conformational assignments.

Both the WT and the Asn5*Asp mutant of the CD4 have shown previously the same or very similar binding activity towards the seven (7) different monoclo- nal antibodies used. Our present finding, (Table 6),

2. Sz&ely et al.iJournal of Molecular Structure (Theochem) 367 (1996) 159-186 183

Table 20

Energetics of hydrogen bonded complex (IA and IB) and ‘salt bridge’ complex (IL4 and IIB) formation according to Fig. 9

Total energy (Hartree) or relative energy (kcal mol“)

6-31G 6-31 + G”

IA E(R) - 283.6339546 - 283.7992789

E(P) - 283.8344272 - 284.0039319

AE(P-R) - 125.78 - 128.40

IB E(R) - 322.661372 - 322.8438879

E(P) - 322.8700548 - 323.0557656

AE(P-R) - 130.93 - 132.93

IIA E(R) - 392.5458715 - 392.7599103

E(P) - 392.6985984 - 392.9233092

AE(P-R) - 95.82 - 102.52

IIB E(R) - 431.5732891 - 431.8045193

E(P) - 431.734226 - 431.9751429

AE(P-R) - 100.97 - 107.05

AE. (kcal mo1) and d(A) E (Hartree) and d(A)

Different pre-orientation Bronsted complex - optimized geometry

Level of theory 0” 45” 90”

HF/3-21G AE 24.069938 - 73.281863 - 129.27915 - 429.5598529

d 3.8 3.8 3.8 3.824488

which shows that the conformation at LYS~~ and Asp5* in the Asn5’Asp mutant has conserved the conformation of the wild-type Lys& and Asn52, is in agreement with the above biological binding activity. Otherwise the conformation of the Pro48-Ser49-LysSo segment of the mutant became more rigid due to the LYS~~-- Asp52 hydrogen bonding but it more or less retained its conformation.

Changing the Phe43 of the WT to Ala, leading to the Phe43Ala mutant, made backbone conformation more rigid at position 43 than that of the WT. Changing the Phe43 of the WT to Ile or Leu, leading to Phe4311e and Phe431eu respectively, left the backbone conformation at position 43 practicly as flexible as in the WT.

Ab inito MO conformations have shown that the - NH3+...- OOC ‘salt-bridge’ has changed to a hydrogen bonded neutral complex, -NH2. * .HOOC-, while the ionic salt bridge prevailed in the ion pair, - C(NHz)+... -OOC-, complex form.

We were able to explain, on the basis of a combined computational model, several structural characteris- tics of the CD4-gp120 complexes. With this approach we could interpret the conformational and electro-

static factors of CD4, in spite of the missing 3D structure of gp120. Our newly established model might serve a good basis for rational drug design of the CD4-gp120 binding inhibitors.

Acknowledgements

The continuous financial support of the Natural Sciences and Engineering Research Council of Canada and the financial supports of the Hungarian Science Foundation (OTKA F13060, TO17192 and F013799), as well as a grant for computational equip- ment provided by the Higher Educational Develop- ment Foundation (FEFA HU-525) are gratefully acknowledged. Research was supported in part by the National Cancer Institute, DHHS, under contract with AHL. The content of this publication does not necessarily reflect the views or policies of the Depart- ment of Health and Human Services, nor does mention of trade names, commercial products or organization imply endorsement by the US Govern- ment. One of the authors (Zolt8n Szekely) is grateful

184 2. S&ely et al.t.lourrtd of Molecular Structure (Theo&em) 367 (1996) 1.59-186

I I I I I I I I I 2 4 6 8 IO

DISTANCE ii,

Fig. 10. Energy lowering during complex formation between protonated amine and acetate ion. The different erossections of the potential energy surface suggest that indeed there is a pre-orientation process.

2

Q, -429.2 Ir 5 .c V

6 ii.5 15 -429,4-

0 90°(perpf2ndiculor 1 H4

I t d

-429.61 1 I I I I I i I I 1 2 4 6 8 IO

Distance (ii) Fig. 11. Energy lowering during the complexation between protonated guanidine and acetate ion. The different erossections of the potential energy surface suggest that indeed there is a pre-orientation process.

2. Szt%ely et aLlJournal of Molecular Structure (Theochem) 367 (1996) 159-186 185

p2 CHg

/

Fig. 12. A schematic illustration for the aromatic lock involving

Tr#*’ and Tyr 4’S of the gp120 and Phe43 of CD4 showing the

Lewis complex between Trp43s and Phe43 as well as the Bronsted

complex between Tyr435 and Phe43:

for an Eotviis Fellowship granted by the National Scholarship Board of the Ministry of Culture and Edu- cation as well as for a fellowship provided by the Word Bank (WO15835).

Research sponsored in part by the National Cancer Institute, DHHS, under contract with ABL. The con- tents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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Documents

Suggested binding mechanism of the HIV-gp120 to its CD4 receptor