A new method for the interpretation of dynamics trajectories in the conformational analysis of HIV receptor mutants

Journal of Molecular Structure (Theochern) , 286 (1993) 165- 182

0166-1280/93/$06.00 0 1993 - Elsevier Science Publishers B.V. All rights reserved 165

A new method for the interpretation of dynamics trajectories in the conformational analysis of HIV receptor mutants

Zolth Szikely **a Andris Perczelb, Botond Penkea, J6zsef Molmir” ,

aDepartment of Medical Chemistry, Albert Szent-Gycrgyi Medical University, Ddm te’r 8. H-6720, Szeged, Hungary bDepartment of Organic Chemistry, Lo’rcind E&v& University, P&m&y PPter S&.iny 2. H-1518, Budapest, Hungary ‘Department of Microbiology, Albert Szent-Gykgyi Medical University, Ddm te’r 10. H-6720, Szeged, Hungary

(Received 17 May 1993; accepted 18 June 1993)

Abstract

Several mutants of the CD4 receptor of the cell membrane surface protein of the human T-lymphocytes were shown to have reduced binding potential for the gpl20 envelope glycoprotein of human immunodeficiency virus (HIV). Simultaneously, the loss of its original immunological activity, the ability to recognize. the major histo- compatibility complex class II (MHC II) antigens, has also been observed. Only a single modification of the CD4, the Asn52Asp mutant, resulted in a noticeable functional separation of the two different activities: the loss of its gpl20 binding and the preservation of its original MHC II immunological activity. Although a large variety of point mutants have been generated, no consistent model has been suggested which could offer an explanation or a structure-activity relationship of the CD4 receptor. In this paper a generalized model is provided on the basis of selected, well established, mutants. Of the numerous mutants, published in the literature, three were selected for the present study where two of them were mutated at position 52 (Asn52Asp and Asn52Ala) and one at position 46 (Lys46Ala). The highest resolution X-ray geometry of the wild-type CD4 receptor has been used for the molecular dynamics (MD) simulations. The conformational behaviour of the wild-type and several of its mutants were investigated using an empirical force field (AMBER) both in terms of gradient geometry optimization and MD simulations. The global character of the backbone conformation was generally preserved during MD simulations. By contrast, the 41-60 subunit of the Lys46Ala mutant showed a significant modification.

Trajectory analysis of MD simulations (using 50 ps time intervals) of the wild-type CD4 and its three different mutants were performed using a new type of trajectory interpretation. This involved the use of the amino acid conformation assignment of proteins (ACAP) software which has been developed by using ab initio-type calculations on model peptides, for the notation of the secondary structure. This method has been adapted as a convenient tool for the analysis of dynamical trajectories.

Introduction

Acquired immunodeficiency syndrome, (AIDS)

is caused by a retrovirus known as human immuno-

deficiency virus (HIV). In the infected patients the

*Corresponding author.

activity of a subgroup of the peripheral T-

lymphocytes decreases. These special T-cells pos-

sess a cell membrane surface glycoprotein, called

the CD4 receptor [1,2]. The CD4 molecule, as a

membrane protein, serves as specific receptor for

HIV. This molecule consists of over 400 amino

acid residues. The first (Dl) and second (D2) extra-

166 Z. SzPkely et al./J. Mol. Struct. (Theochem) 286 (1993) 165-182

II CD4

Extracelhlar region (371 amino acid residues)

Membrane region (24 amino acid lesidoes)

Inttacelhdar region (38 amino acid residues)

Fig. 1. A schematic structure representation of the full CD4 membrane glycoprotein molecule of the T-cell. The four extracellular immunological domains (Dl, D2, D3 and D4) are clearly shown.

cellular domains of CD4 (see also Fig. 1) were subjected to mutagenesis. The activity of mutants were determined by standard biological techniques [3-l 11. The structure of the DlD2 superdomain of the wild-type (WT) mutant was determined by X-ray crystallography [ 12,131. However, no structure-activity relationship has emerged so far from these studies.

The initial event in the HIV infection involves the interaction of the virus envelope glycoprotein, gp120, with the CD4 receptor, thereby allowing the virus to enter into the human T-cell. Since the HIV infection will be effective if and only if the gp 120 of the virus will bind to the CD4 receptor of the human T-lymphocytes, therefore the prevention of such a binding may be regarded as a possible therapeutic method. Consequently, among the different chemotherapeutic possiblities the selective inhibition of the virus binding procedure has been recently investigated. As a binding requires two components (the CD4 receptor as well as the gp120 of the virus), therefore covering a relevant surface portion of one or the other component may lead to stop the infection cascading.

One of the two alternatives involves the covering of the active site of the CD4 receptor [14-161. However, care must be applied in designing such

Scheme 1 The variation of binding activity with mutation

Mutants of CD4

Wild-type Lys46Ala Asn52Ala Asn52Asp

Binding activity

mAb HIV gp120

full 100% reduced 5% full 110% full 24%

a cover because the CD4 receptor is a multi-functional entity. This can be clearly seen from the fact that the main role of the CD4, in the normal phy- siology, is a receptor function for the major histo- compatibility complex class II (MHC II) antigens. The lack of such an interaction would result in a fatal physiological defenselessness. Therefore any covering, which aims to prevent the gp120 binding to CD4, should not inhibit the binding of the MHC II molecules to CD4. There are two biological testing methods for the determination of MHC II binding activity of CD4 or its mutants. The more recent method is the “rosette formation” technique [ 171 which is more direct and perhaps a more quan- titively known example for the separation of the two types of biological activity. (The preservation of the MHC II binding activity is a crucial requirement for any type of CD4 mutant to be considered as a normally functioning receptor.) On the bases of this remarkable experimental result (Scheme l), a working hypothesis was established as shown in Fig. 2, which provides a simple explanation of the binding affinity. We believe that the transformation of Asn to Asp at position 52 induces directly the binding inhibition of the gp120 and may also cause immunosuppression by the simultaneous inhibition of the MHC II recognition site. For the design of suitable inhibitor molecules one needs high resolution X-ray or NMR structural data of the CD4 receptor.

The other alternative approach for the inhibition of the binding of gp120 to the CD4 is the covering of the active region of the gp120 surface [18]. Since, at the present time the real conformation of the gp120 is unknown, therefore the geometrical

2. Szikely et al.lJ. Mol. Struci. (Theochem) 286 (1993) 165-182 161

Wild type Normal gP 120

binding

Fig. 2. A schematic representation of the gp120-CD4 binding theorem in the case of wild-type (top) and for its Asn52Asp mutant (lower part). Arrows point from the proton donor towards the proton acceptor.

properties of its active site for gpl20-CD4 complex formation is also unknown. Furthermore, the design of an inhibitor is totally based on the similarity of a suitable structural motif of the Dl domain in CD4 which plays a major role in gp120-CD4 binding.

A mapping of the active region of the CD4 receptor systematic point mutation, in the region invol- ving the 19th-89th amino acid residues, has been performed. Most of these mutations involved the alanine scanning mutagenesis technique, when more complicated side-chains were changed to the simpler methyl group. The primary target of mutation was the 40-55 segment of Dl. Subsequent testing of the biological activity of the mutants was performed to assess the role of a given amino acid, at a given site. A number of mutants have shown either a significant drop or practically no change of both types of biological activity. Since our primary purpose is to find a connection between the protein structure of the CD4 receptor

and its gp120 binding activity, a number of mutants had to be investigated where the replacement of amino acid took place at strategic positions. Site 52 has been already proven to be important, as discussed above, therefore the Asn52Ala was investigated in the present study in addition to the Asn52Asp. The other important site was Lys46. When Lys was changed, via alanine scanning mutagenesis, then the resultant Lys46Ala molecule has practically lost its gp120 binding ability and its monoclonal antibody (mAb), binding potential was also reduced. Since this latter factor is frequently considered to be diagnostic of conformational change therefore the Lys46Ala mutant, which showed a substantial drop of its biological activity, was interpreted to suffer a significant conformational change. This suggested that site 46 must be strategically important. Scheme 1 shows a summary of the biological activity of the mutants studied in this paper.

Dozens of mutants prepared by cloning showed that the variation of the amino acid sequence can not result in a noticable separation of the two types of biological functions. Fortunately, in the case of an enzymatic replacement of Asn at position 52 by Asp (equivalent to the Asn52Asp mutant) the two biological effects were separated. In this case the gp120 binding activity is reduced from 100% to 24%, but the binding activity of conformational dependent monoclonal antibodies was totally preserved [19].

Consequently, the Asn52Asp mutant is expected to provide the most valuable information about the CD4 binding site. Such an understanding is a prerequisite for any design of selective binding inhibitors.

scope

Structural hypothesis for the separation of dual

biological functionality

At the pH level of blood (pH 7.4) all the amino and guanidino groups of the side-chains in lysines and arginine are fully protonated. Since the CD4

168 2. Szhkely et al./J. Mol. Struct. (Theochem) 286 (1993) 165-182

molecule is positively charged, therefore one can assume that the surface of gp120, at least at the binding region, is negatively charged. This is further corroborated by the fact that the gp120 itself cannot bind to the CD4 when the positively charged Lys or Arg are eliminated. This is clearly observed when the strategic positions Lys29, Lys35, Lys46 and Arg59 are changed to alanines

[lll. The Asn52Asp mutant is the only known

example for the separation of the two types of biological activity. (The preservation of the MHC II binding activity is a crucial requirement for any type of CD4 mutant to be considered as a normally functioning receptor.) On the basis of this remarkable experimental result (Scheme l), a working hypothesis was established as schematically shown in Fig. 2, which provides a simple explanation of the binding affinity. We believe that the transformation of Asn to Asp at position 52 induces a direct reorientation of the Lys46 side- chain, namely that the intermolecular charge trans- fer type interaction between gp120 and CD4 will be replaced by the formation of an intramolecular (-NH;. . .- OCC-) salt-bridge, without a drastic backbone modification. More specifically this model presumes, that the original secondary structure of the {41,60} region of CD4 is preserved in the Asn52Asp mutant, as the MHC II molecules can still bind to this mutant of the CD4 as repre- sented by mAb binding activity (see Scheme 1).

The manifestation of such a hypothesis during a MD simulation is that no major conformational change is expected to occur for the wild-type or for the Asn52Asp mutants (changed to Ala or Asp) of CD4.

Computational ver@cation of the structural

hypothesis

The verification of the above working hypothesis was carried out by computational methods on the DlD2 and Dl fragments of CD4. Because of the limitation of todays computers only empirical approaches (force fields) can be used for the total

energy calculation of such an extended molecular system (over 1500 atoms). Molecular mechanics (MM) gradient optimization method has been applied to the AMBER force field of DlD2 and Dl while the molecular dynamic (MD) simulation has been applied to the Dl fragment only.

The dynamic behaviour of different mutant proteins during MD simulations can give several important structural data. Using a classical type of trajectory analyses for such an extended system investigated over 5Ops, it is almost impossible to extract data on conformational changes. On the basis of atomic velocity, over the time average dynamical behaviours, peptides or protein fragment can be investigated. However, the familiar trajectory analysis of different internal coordinates can be very lengthy and information overflow of MD simulations can be confusing. For this reason, a universal notation which could simplify the interpretation of such MD simulation results had to be interpreted.

The 49 different relaxed conformations of For- Ala-Ala-NH2, calculated previously by the ab initio method (SCF 3-21 G), incorporate 2 x 49 = 98 subconformations, resulted in conformations where the combination of only the nine basic subconformations (cQ,o!L, P L,yo,yL, 6 s o, L, eo and Ed) were observed [20-221. According to the essential backbone conformational types the clas- sification of the backbone torsion angles dis- tributed over more than 11000 amino acids incorporated in selected proteins with X-ray determined structures was reported previously. The application of these located minima as “conformational centres” for secondary structure descrip- tion of globular proteins was already found suitable [20]. The conformation alterations during the dynamics of a backbone fraction (41-60) of the CD4 protein have been analysed here using the nine basic conformation types.

Methods

The input geometry of the Dl and D2 domain of the CD4 and its fragment were created by the

2. Szt!kely et al.lJ. Mol. Struct. (Theochem) 286 (1993) 165-182 169

INSIGHT II 2.1.0 program [23] on the basis of the Brookhaven Protein Data Bank file pdblcd4 and file pdb2cd4. Hydrogens were added to heavy atoms automatically according to the AMBER force field [24]. The molecular geometry optimization and MD simulations were performed using the AMBER force field potential within the DISCOVER 2.8 [23] program on an IBM RS6000/320H work- station. Gradient geometry optimization was performed on both crystal structures, while the MD simulation was carried out on the higher resolution (2.3 A) crystal structure (pdblcd4).

The following options were used for calculations. A (1) standard charge set from the AMBER “all atom” force field, with (2) distance dependent dielectric constant with a (3) 10 A cutoff of the non-bonded interactions were applied. Dur- ing structure optimizations (4) a conjugate gradient algorithm was used with a 0.001 kcalmol-’ A-’ derivative convergence criteria. At (5) 3 10 K equi- libration temperature, the following options were used: 1 fs time step, 50~s total time, recompute neighbour list of residues every 20 steps and his- tory output every 50 steps for dynamic simulations.

The MD simulation resulted in Cartesian coordinates which were converted to backbone torsion angles (b,+) according to IUPAC-IUB convention. These torsion angle pairs were then assigned to one of the nine “conformational centres” as described elsewhere [20-221. The codes and the locations of the nine “conformational centres” are summarized in Table 1. The amino acid conformation assignment of proteins AcAP software [20] has been used to analyse the conformational change of the 41-60 backbone segment of the Dl domain during 50~s change of the MD analysis.

Results and discussion

Molecular structures optimization of the DID2 and

Dl domains

The global structure of the extracellular region of the CD4 receptor (Fig. 1) can be partitioned into two superdomains (DlD2 and D3D4) where both

of them can be further partitioned into two addi- tional domains (Dl and D2 as well as D3 and D4 respectively). The fragment containing the first two domains (DlD2) were biotechnologically pro- duced, crystallized and subsequently analysed by X-ray crystallographic methods in two independent laboratories [ 12,131. The connecting segment between second (D2) and third (D3) domains of CD4 is a flexible short amino acid sequence (also shown in Fig. 1) which allows therefore the trunca- tion of the overall CD4 protein, fragmenting off the DlD2 portion. This DlD2 superdomain incorpo- rates a total of 183 amino acid residues. This fragment seems to have a major role in the binding of the gp120 as well as the MHC II molecules. It is reasonable therefore to assume that this is a desir- able size model for structure investigation. The question whether the DlD2 superdomain is desir- able and the D 1 domain is the minimum size model to be investigated has not been answered at the start of this work. In fact, one of the aims of the present research was to find an answer to the above question.

The available two X-ray data files of the DlD2 fragment were compared on the basis of secondary structures. Using our linearized secondary structure notation, certain conformational differences were observed (see Table 2) for the 95 amino acid residues, between Lys2-Leu96, which is the assign- able portion of the 97 amino acid residues contain-

Table 1 Average torsion angle (+,$J) values of the basic backbone conformation types or “conformational centres” obtained by averaging For-(Ala)z-NHz computed structures at the HF/3-21G level of theory

Conformational type

ffL -68.6 -17.5 CyD 61.8 31.9 PL -167.6 169.9 YL -84.5 68.7 -YD 74.3 -59.5 6L -126.2 26.5 5D -179.6 -43.7 EL -74.7 167.8 ED 64.7 -178.3

170 Z. Szkkeiy et al./J. Mol. Slruci. (Theo&em) 286 (1993) i65-182

X-ray determined MM computed

Scheme 2. Number of coincidences in the 95 confo~ation assi~ment for amino acid No. 2-96 in the Dl domain.

ing the Dl domain. Nevertheless, out of the total 95 residues assigned (see Scheme 2) 55 residues have identical backbone conformations while 40 residues have different backbone conformations as dete~ined by ACAP PO]. The X-ray measure- ments were performed in two independent laboratories at the same time, therefore these structural data do not contain any predetermined structural motifs. For this reason the comparison of the two X-ray data files were extremely important.

In our investigation, first of all the minimized structure ,of DlD2 (Fig. 3A) were compared to its truncated derivative labelled as Dl (Fig. 3B). On comparing the amino acid residues no major conformational differences were found between the isolated Dl and the Dl as part of DlD2. The comparison of the X-ray data and the two MM optimized structures (DlD2 and Dl separately) legitimized the separation of the Dl domain from the Dl D2 superdomain fragment (see Table 3). In the view of the 95 - 55 = 40 conformational differences, assigned between the two X-ray structures, a more rigorous similarity of the two computed Dl parts was established (see Scheme 2).

In fact the conformations of 72 amino acid residues out of the 9.5 agreed when the Dl optimized structure is compared with the Dl portion of the optimized DID2 superdomain. The degree of similarity of the two X-ray structures (57.9%) is smaller than the one computed for the two minimized Dl structures (75.8%). The resemblance between the high resolution (2.3 A) X-ray structures and any of the calculated conformations is shown by the values 61 .l%, 63.2% for DlD2 and Dl respectively. Therefore the influence of D2 on Dl can be ignored at least in a preliminary structure investigation,

Molecular dynamics simulations of the DI domain

The conformational properties such as selected intramolecular distances and backbone torsion angles of Dl of CD4 (Fig. 3B), which consists of 97 amino acid residues, were analysed first by trajectory analyses. By monitoring the atomic motions, structural data were obtained in terms of trajectories (distances and torsional angles) after simulations over a time period of 50~s.

2. Szekely et al./J. Mol. Struct. (Theochem) 286 (1993) 165-182

Table 2

171

Torsion angles (4, $), conformational assignment codes (C) and deviation (Dev) from the standard “conformational centres” for the two X-ray structures (2cd4 and lcd4) as well as torsional angle differences (A4, A$) between the two X-ray structuresa

No. AA 2cd4 Resolution: 2.4 A

4 i

lcd4 lcd4-2cd4 Resolution: 2.3 A

C Dev 4 * C Dev A+ A+

LYS -93.01 VAL -127.21 VAL -125.83 LEU - 127.02 GLY -137.60 LYS - 109.72 LYS -43.07 GLY 87.51

10 ASP - 100.06 THR -125.74 VAL -109.11 GLU -131.17 LEU -97.29 THR -117.30 CYS -149.60 THR -131.09 ALA - 129.99 SER -53.98

20 GLN -100.82 LYS -65.98 LYS -170.86 SER -80.90 ILE -89.21 GLN -115.09 PHE -139.28 HIS - 146.98 TRP -108.89 LYS -108.86

30 ASN -98.20 SER -71.93 ASN -74.28 GLN 51.57 ILE -60.38 LYS -81.86 ILE -97.90 LEU -168.71 GLY -179.43 ASN -110.45

40 GLN -98.15 GLY 54.16 SER -93.65 PHE -90.29 LEU -100.72 THR -109.56 LYS -98.63 GLY -70.22

163.41 er 18.83 -10.85 141.67 eL 130.09 pJ_ 56.71 - 122.38 132.53 p,_ 147.13 /3r_ 47.57 -125.13 146.44 /?IL 137.78 flL 51.75 -118.22 105.09 TL 177.69 pL 30.99 -94.52 165.38 eL 135.03 Et_ 47.96 -117.00 131.58 eL 125.76 E,_ 52.61 -40.51 145.75 q -3.44 cxo 43.70 53.53 19.11 oo

-164.07 q 37.87 - 110.59 175.22 et, 128.82 pL 58.65 -101.25 132.11 eL 163.79 et_ 34.64 -113.37 138.67 cL 109.21 -yL 61.80 -122.65 111.54 “/L 158.81 E,_ 24.31 -97.57 134.50 EL 154.14 EL 44.74 -96.59 144.76 eL 128.67 pL 44.99 -150.05 111.38 flL 145.83 pL 43.73 -123.19 100.13 -yL 155.12 /JL 40.41 -105.44 65.80 yL

-32.65 (Ye 10.28 -55.24 138.58 eL 108.64 yL 43.15 136.56 147.62 pL 75.60 yL 19.76 -65.04 93.38 “1~

158.03 pL 12.31 - 100.76 160.31 eL 78.68 -yL 10.61 -89.39 102.09 yL

-128.81 eL 65.03 -75.78 58.23 yL 115.46 yL 55.88 -91.75 108.64 yL 154.65 pL 32.16 -153.83 165.08 pL 162.53 pL 21.90 -156.55 165.95 pL 117.18 “iL 54.27 -112.69 138.93 eL 167.09 eL 34.17 -137.40 169.10 /3r

-171.73 EL 31.17 -106.67 163.12 E,_ -27.95 (Ye 9.26 -70.32 89.68 yL

19.10 q 47.73 -178.11 -110.46 So 4.34 ao 29.40 172.88 35.21 SL

128.52 tr 41.81 -94.88 113.19 yL 136.57 eL 32.04 -72.29 125.43 eL

-48.55 oL 41.17 -94.26 -45.14 (YL 164.27 pL 5.74 -165.56 139.15 pL 179.78 pL 15.41 -141.20 -178.35 pL 131.98 cL 50.61 - 107.08 119.01 ÎL 102.68 -rL 36.62 -121.38 97.29 7L

-85.95 or, 33.24 89.08 -107.29 yt, -48.38 (Ye 37.50 -130.24 15.20 6L 175.46 eL 17.37 -125.85 -178.34 pL 126.56 eL 48.76 -106.50 125.05 cL 121.14 ail 58.12 -113.84 118.01 “/L 114.96 ail 48.37 -113.59 176.14 eL 173.03 EL 6.89 53.80 -167.16 E,,

68.99 82.16 58.66 4.83 48.52 0.79 49.61 8.80 19.97 43.08 55.69 -7.28 40.68 2.56 15.23 -34.18 36.65 -10.53 44.48 24.49 48.41 -4.26 57.36 8.52 40.40 -0.28 31.78 20.71 61.09 -0.45 49.85 7.90 21.14 24.55 35.11 - 1.26 60.12 237.38 31.43 0.98 27.12 70.10 33.75 -8.49 13.63 13.43 40.59 23.34 14.59 - 14.55 11.73 -9.57 47.71 -3.80 30.21 -28.54 32.31 -8.47 25.32 1.61 66.78 - 103.83 61.54 121.31 45.68 -34.50 42.44 9.57 36.30 3.64 30.82 3.15 28.90 38.23 55.14 3.37 46.66 -23.23 50.02 -20.92 12.00 -36.59 43.37 -35.56 53.28 -5.78 57.38 -4.28 39.77 -14.96 15.59 124.02

-21.74 2.44

-0.69 -32.69 -12.31

-3.45 19.99 22.51

339.29 260.93 -25.12

2.33 -24.3 1

-9.38 -17.29 -45.70 -89.32 171.23 38.98 17.78 2.28

23.41 187.04 -6.82 10.43 3.42

21.75 2.01

334.85 117.63

-129.56 30.87

-15.33 -11.14

3.41 -25.12

-358.13 -12.97

-5.39 -21.34 -63.58

-353.80 -1.51 -3.13 61.18

-340.19

172 Z. Szikely et al./J. Mol. Strut. (Theochem) 286 (1993) 165-182

Table 2 (continued)

No. AA 2cd4 lcd4 1 cd4-2cd4 Resolution: 2.4 A Resolution: 2.3 A

4 1c, C Dev c ?I, C Dev Ati W

PRO -71.56

SER -152.81

50 LYS -67.58 LEU -89.08

ASN -52.82 ASP -81.55

ARG -136.28 ALA -88.01

ASP -169.78 SER -136.81

ARG -122.35 ARG -74.02

60 SER -54.04

LEU -101.08 TRP -61.93

ASP -67.06 GLN -94.86

GLY 92.28

ASN -125.94 PHE -130.55 PRO -73.19 LEU - 104.47

70 ILE -112.70 ILE -119.53 LYS -100.49

ASN 31.67 LEU -56.93 LYS 135.15

ILE -72.02 GLU -72.31

ASP -80.30 SER -74.10

80 ASP -174.50 THR -95.32 TYR - 114.84

ILE -128.62

CYS -124.87

GLU -111.93 VAL -132.40

GLU 55.75 ASP -102.74

GLN -114.37 90 LYS -118.40

GLU -131.09 GLU -91.35

VAL -124.52 GLN -77.70

114.75 ^1L 167.19 pL

-19.10 QL 7.39 6L

-52.26 (Ye 16.40 bL

-20.71 bL

156.41 eL

167.91 pL -173.12 /JL

114.55 Îr.

-34.96 (Ye -19.87 (Ye

-6.50 dL -33.43 (YL -24.09 CXL

7.44 6L 28.01 (Yo

131.06 pL

74.95 ÎL 149.37 EL 128.79 cL 125.13 cL 121.66 car.

170.98 eL

$4.30 (Yo 153.54 .zL 153.95 pJ_

-24.79 aL -4.91 q,

2.11 cq 125.30 eL

171.08 pL 133.09 .zL

147.82 cL

143.93 ,& 118.99 car.

133.73 EL 112.72 -yL

-124.44 e,-, 33.26 SL

129.90 .zL 135.73 q_ 120.89 pL 138.62 eL

109.18 +yL 132.04 eL

47.83 -75.37 27.76 car. 41.95 -3.81 -86.99 15.04 -91.26 175.72 cL 18.36 61.55 8.53 9.46 -58.61 -26.70 (Ye 4.12 8.97 -7.60

41.75 -97.85 12.46 SL 31.64 -8.77 5.07 26.94 -63.70 -49.02 CYL 21.84 -10.88 3.24 45.78 -86.23 -5.35 cq_ 32.11 -4.68 -21.75 48.27 -105.42 -25.01 ozL 42.78 30.86 -4.30 17.52 -94.65 151.91 q 25.50 -6.64 -4.50 2.95 -164.36 177.60 ,!?L 8.35 5.42 9.67

35.16 -147.18 172.18 pL 20.55 10.37 345.30 59.45 -112.85 92.97 -& 37.32 9.50 -21.58 13.72 -66.95 -34.24 cxL 8.22 7.07 0.72 11.35 -55.44 -18.38 ‘I~ 11.42 -1.40 1.49 41.47 -98.39 -29.66 (Ye 35.77 2.69 23.16

6.28 -48.55 -33.78 ffL 15.61 13.38 -0.35 5.36 -65.54 -11.50 QL 15.95 132.6 12.59

36.68 -107.52 -1.92 6, 34.01 -12.66 -9.36 30.73 86.47 17.21 (Yo 28.71 -5.81 -10.80 56.96 -108.19 121.98 eL 56.75 17.74 -9.08 46.47 -131.50 76.81 car. 47.69 -0.95 1.86 18.49 -74.78 153.01 .zL 14.79 -1.59 3.64 49.07 -111.60 119.45 yL 57.53 -7.13 -9.34

57.14 -98.26 111.29 Îr. 44.76 14.44 -13.84

63.50 -106.31 117.65 +yL 53.59 13.22 -4.01

25.99 -79.13 -61.52 oL 38.05 21.36 -232.5

44.24 -118.20 76.18 ^/L 34.52 -149.87 11.88 22.78 -30.61 110.50 yL, 68.20 26.32 -43.04 36.16 -105.04 151.06 eL 34.65 30.11 -2.89

9.63 -70.84 -2.39 (YL 26.11 -0.82 22.40 24.27 -81.29 -25.78 (Ye 18.64 -8.98 -20.87 34.19 -67.84 -7.33 CYL 20.52 12.46 -9.44 42.50 -64.03 112.26 yL 48.13 10.07 -13.04

7.00 170.97 178.28 pr. 23.01 345.47 7.20

40.37 -85.90 121.89 eL 47.26 9.42 -11.20 44.84 -98.40 169.79 Ed 23.78 16.44 21.97

46.84 -158.26 126.55 ,BL 44.34 -29.64 -17.38 64.49 -110.08 129.42 q 52.20 14.79 10.43

50.47 -124.28 106.06 q‘L 54.57 -12.35 -27.67 65.06 -116.55 112.84 Î~ 54.55 15.85 0.12

54.60 50.28 -98.31 ,yD 45.64 -5.47 26.13 24.41 -100.72 -19.27 (YL 38.84 2.02 -52.53 54.86 -82.37 128.84 eL 39.71 32.00 -1.06 54.20 -107.53 162.44 cL 33.26 10.87 26.71 61.11 -159.53 86.27 SL 68.43 -28.44 -34.62 33.60 -70.57 122.15 eL 45.84 20.78 -16.47 56.92 -125.75 115.65 n 62.50 -1.23 6.47 35.89 -89.31 118.14 ant 49.67 -11.61 -13.90

Z. Szbkely et al./J. Mol. Struct. (Theochem) 286 (1993) 165-182 173

Table 2 (continued)


4 *


C Dev d 11, C Dev A+ A*

LEU - 109.45 LEU -113.85 VAL -113.58 PHE -141.04 GLY -144.47

100 LEU - 144.98 THR -117.04 ALA -65.08 ASN -78.87 SER -163.13 ASP 43.33 THR -96.86 HIS 49.46 LEU -78.98 LEU -107.48

110 GLN -45.07 GLY 97.46 GLN -71.97 SER -76.67 LEU -132.89 THR -125.18 LEU -122.71 THR -129.84 LEU -89.30 GLU -92.37

120 SER -134.49 PRO -77.91 PRO -61.91 GLY -61.81 SER 171.72 SER -131.35 PRO - 104.38 SER -141.45 VAL -96.62 GLN -106.34

130 CYS - 172.88 ARG -121.50 SER -62.50 PRO -55.02 ARG -120.20 GLY 89.81 LYS -47.51 ASN -142.29 ILE -89.25 GLN -135.65

140 GLY 165.08 GLY -127.33

121.16 car 58.09 -92.35 121.29 tL 49.75 17.10 0.13 122.81 q 59.64 -110.33 120.46 yL 57.85 3.52 -2.35 143.99 CL 45.59 -117.05 137.10 EL 52.31 -3.47 -6.89 155.31 /?L 30.30 -139.86 160.69 pL 29.23 1.18 5.38 164.84 pL 23.68 -132.75 158.69 pL 36.61 11.72 -6.15 115.07 pL 59.31 - 149.02 95.77 -yL 69.97 -4.04 -19.30 146.15 q 47.55 -110.54 176.41 cL 36.86 6.50 30.26 88.85 -yL 27.98 -114.27 159.44 EL 40.44 -49.19 70.59 36.23 -y,_ 32.95 -74.97 -50.13 aL 26.01 3.90 -86.36

-126.20 pL 64.06 -142.52 152.60 /IL 30.47 20.61 278.80 32.84 aD 18.49 -44.19 101.81 “IL 52.16 -87.52 68.97

120.70 eL 52.05 -43.12 84.45 -yL 44.28 53.74 -36.25 70.11 oo 40.15 -106.18 73.55 -yL 22.22 -155.64 3.44

132.04 eL 36.02 -45.95 116.15 eL 59.11 33.03 -15.89 138.49 q 43.97 76.35 167.35 co 18.48 183.83 28.86 137.95 q 42.06 64.70 124.21 e,, 57.49 109.77 -13.74

-24.57 ÎD 41.91 78.75 -90.33 ÎD 31.15 -18.71 -65.76 154.86 cL 13.22 -42.91 171.17 q 31.97 29.06 16.31 173.25 cL 5.80 -122.23 124.31 pL 64.32 -45.56 -48.94 126.08 pL 55.90 -83.18 142.66 q, 26.53 49.71 16.58 147.73 pL 47.86 -148.00 107.49 pL 65.42 -22.82 -40.24 143.52 pL 52.07 -99.66 137.88 eL 38.96 23.05 -5.64 139.75 pL 48.32 -109.41 162.81 cL 35.07 20.45 23.06 131.17 q 39.43 -121.95 124.51 eL 64.08 -32.65 -6.66 123.58 eL 47.62 -93.33 131.56 eL 40.75 -0.96 7.98 153.22 /IL 37.07 -149.89 145.93 pj_ 29.80 -15.40 -7.29 145.75 CL 22.28 -44.84 150.98 eL 34.27 33.07 5.23

-13.72 rxL 13.50 -48.86 -22.95 cq 14.48 13.05 -9.23 -17.03 (YL 10.21 -113.67 11.76 bL 19.35 -51.86 28.79 158.14 pL 23.79 -124.09 128.94 fiL 59.76 -295.81 -29.20 141.83 pL 45.85 -135.14 61.67 6, 36.29 -3.79 -80.16

-155.54 q 47.17 -68.46 -171.23 cL 21.88 35.92 -15.69 116.33 pL 59.61 -110.75 146.61 cL 41.82 30.70 30.28 131.81 cL 42.14 -120.95 169.58 q 46.28 24.33 37.77

-177.62 q 34.84 -172.26 96.47 ,& 73.58 -65.92 274.09 134.10 /?L 36.19 -94.90 144.19 q_ 31.07 77.98 10.09 124.85 cL 63.52 -132.80 122.32 pL 58.95 -11.30 2.53 152.06 eL 19.91 -65.26 176.73 eL 12.99 -2.76 24.67

-24.44 cxL 8.16 -64.12 83.58 Î~ 25.23 -9.10 108.02 46.47 S, 20.85 76.36 150.71 co 33.11 196.56 104.24

-42.61 “1D 22.93 61.73 -129.60 co 48.79 -28.08 -86.99 147.27 eL 34.07 20.40 61.07 (Yo 50.64 67.91 -86.20 131.64 pL 45.87 -75.18 121.50 eL 46.30 67.11 -10.14 164.07 EL 15.02 -118.63 127.17 .zL 59.84 -29.38 -36.90 164.72 &, 32.37 -128.72 165.90 & 39.09 6.93 1.18

-175.69 cD 100.41 179.87 -151.63 6, 107.93 14.79 24.06 -2.58 6L 29.10 -120.11 -77.02 6, 68.19 7.22 -74.44

174 2. Wkely et al.lJ. Mol. Struct. (Theochem) 286 (1993) 165-182

Table 2 (continued)


4 li,


C Dev Q, II, C Dev Ah9 W

LYS THR

LEU SER VAL

SER GLN LEU

150 GLU LEU

GLN ASP

SER GLY THR TRP

THR

CYS 160 THR

VAL LEU

GLN ASN GLN LYS

LYS VAL

GLU 170 PHE

LYS

ILE ASP ILE

VAL VAL

-170.74 -118.39

-138.12 -137.26 -118.41

-77.54 65.42

-101.56 -86.57 -66.70

-66.12 -90.05 -74.43

126.05 -102.19

-93.75

- 124.20 -110.98

- 120.70 -105.03

-108.73 -152.98

29.93 69.22

-108.22 -143.48 -110.92 -133.00

-112.23 -114.81

-98.73 - 124.94 -137.58 -123.57

-

-45.66 So 9.07 145.09 q 49.24

136.65 ,BL 44.44 143.54 /?L 40.19 98.95 car. 45.44

136.60 q 31.33 97.23 (Yo 65.43

132.88 q 44.06 171.65 q 12.48

-29.65 cxL 4.69 -5.12 cyL 22.34 -8.66 q 33.04 152.50 eL 15.30

,151.52 ED 66.94 117.12 car. 51.55 155.15 EL 22.87 121.74 fiL 64.83 144.42 eL 43.16 120.02 -jy 62.80 127.40 cL 50.52 131.40 eL 49.83 136.22 pL 36.72 55.13 a,, 39.44 0.06 (Yo 32.69

175.30 EL 34.35 143.40 pJ_ 35.83 153.91 EL 38.79 119.57 pL 61.08 130.92 q_ 52.62 124.06 eL 59.35 151.26 eL 29.17 129.65 /?,_ 58.65

156.47 pL 32.89 129.73 pL 59.60

-104.06 -47.01 (YL 45.86 66.68 -1.35 -118.21 137.07 EL 53.27 0.18 -8.02 -119.68 124.14 EL 62.68 18.44 -12.51 -111.36 143.44 CL 44.02 25.90 -0.10 -133.14 75.37 7L 49.10 - 14.73 -49.85

-81.08 117.92 7L 49.34 -3.54 -18.68 86.05 97.16 OD 69.62 20.63 -0.07

-79.32 135.93 EL 32.20 22.24 3.05 -161.85 120.80 PL 49.44 -75.28 -50.85 -170.41 -124.70 PL 65.46 -103.71 -95.05 -151.23 -174.19 PL 22.83 -85.11 -169.07

-37.76 -94.12 OL 71.42 52.29 -85.46 -86.61 37.38 7L 31.39 -12.18 -115.12 -49.05 145.55 CL 33.96 -175.10 297.07

-113.71 102.61 7L 44.76 -11.52 -14.51 -81.57 172.86 EL 8.53 12.18 17.71

-137.14 78.64 6L 53.28 -12.94 -43.10 -76.09 133.27 EL 34.56 34.89 -11.15

-133.06 114.92 PL 64.93 7.64 -5.10 -102.14 132.85 EL 44.43 2.89 5.45 -111.93 157.83 EL 38.54 -3.20 26.43 - 159.46 -140.58 PL 50.18 -6.48 276.80 -66.88 64.27 7L 18.17 -96.81 9.14

76.38 -32.92 7D 26.66 7.16 -32.98 -62.91 172.52 EL 12.70 45.31 -2.78

-159.35 157.19 PL 15.15 -15.87 13.79 -117.52 144.39 EL 48.80 -6.66 -9.52 -135.77 109.63 7L 65.60 -2.77 -9.94

-91.22 145.81 EL 27.50 21.01 14.89 -138.11 91.69 “IL 58.33 -23.30 -32.37

-91.36 141.13 EL 31.45 7.37 -10.13

“4, $, Dev, A# and All, are given in degrees.

The most representative distances (46C”-52C*) of the different mutants resulting from the MD calculations were plotted in all four cases. In the case of the WT and two of its mutants (Asn52Ala and Asn52Asp) the 46C*-52C” distance was preserved relatively constant (6 f 1 A). In contrast, a significant spatial separation, an increase from 5 to

11 A (Fig. 4) of the 46Ca-52CU atoms can be observed in the case of the third mutant: Lys46Ala. The unique dynamic behaviour of the Lys46Ala mutant harmonizes with the conformational changes, indicated by the biological tests as shown in Table 4. In other words the relationship between the loss of all biological activities of the

Z. Szikely et al./J. Mol. Struct. (Theochem) 286 (1993) 165-182 175

Fig. 3. Ribbon models of the CD4 receptor segments and receptor: A, The DlD2 super domain; B, the Dl domain with side- chains of Lys46 and Asn52 explicity shown.

Lys46Ala mutant (Table 4) and the conformational changes measured by the 46C”-52C” spatial separation will lead to the inevitable conclusion that, at least for these four mutants, the biological activity and the conformational stability are inter- dependent in the Dl domain of the CD4 receptor.

In studying the most important mutant, Asn52Asp, the 46N’-52CY distance was monitored in the MD simulation. Figure 5 shows a dramatic reduction of the 46N’-52CY distance from an average of 6A to an average 3 A with respect to the WT mutant. This change indicates the close proximity of the two charged side-chain ends (-NH;. . . OOC-) located at residue 46 and residue 52 respectively. Such a proximity amounts to nothing less than an ion pair interaction or a salt-bridge formation. This salt-bridge makes the

loop more rigid which is clearly seen from the smoothness of the Asn52Asp trajectory plot (lower part of Fig. 5) with respect to that of the WT (upper part of Fig. 5).

In order to obtain a more detailed geometrical picture, the alteration of the backbone torsion angle changes were also monitored for 41-60 residues during the 50~s of the MD simulations. Although different possible analyses can be applied, such as structure overlapping, animation, cluster representation, selected trajectory analysis of internal coordinates etc., we propose here the use of our linearized conformation notation over the 50~s time interval. This method has been applied for the study of two mutants; Lys46Ala which has shown a total loss of biological activity due to its marked conformational change and

176

Table 3

Z. Sdkely et al./J. Mol. Strut. (Theo&em) 286 (1993) 165-182

Comparison of assigned conformations for the two X-ray structures (lcd4 and 2cd4) of DlD2 as well as MM optimized Dl using the two different crystal structures as starting geometries

No. AA lcd4 2cd4 Resolution: 2.3 A Resolution: 2.4 A

X-rav DlD2 Dl Dl DlD2 X-Ray

10

20

30

40

LYS LYS VAL VAL LEU GLY LYS LYS GLY ASP THR VAL GLU LEU THR CYS THR ALA SER GLN LYS LYS SER ILE GLN PHE HIS TRP LYS ASN SER ASN GLN ILE LYS ILE LEU GLY ASN GLN GLY SER PHE LEU THR LYS GLY PRO SER

EL

PL

7L

7L

k h ffD

;;r PL

n n EL

-7L

7L

7L

2 7L CL 7L (YL 7L CL PL CL PL EL R 8, SD CL CL (YL PL PL 7L n %I (YL EL CL EL CL ED Q,L CL

EL

CL

R

7L

CL

PL

EL

7D

CL

CL

PL

YL

EL

EL

PL

PL

EL

(YL

7L

7L

PL

7L

PL

CL

PL

PL

EL

PL

CL

ffL

(YL

(YD

EL

EL

aL

PL

PL

EL

7L

7D

(YL

EL

7L

7L

CL

EL

7L

PL

EL

7L

n n EL

EL

EL

7D CL CL PL 7L 7L EL PL YL CL aL 7L

2 7L EL fL PL PL CL PL EL QL (IL aD EL EL aL PL PL EL YL 7D (YL CL CL 7L CL EL 7L PL

2. Mkely et a/./J. Mol. Struct. (Theochem) 286 (1993) 165-182 177

Table 3 (continued)

No. AA lcd4 2cd4 Resolution: 2.3 A Resolution: 2.4 A

X-ray DlD2 Dl Dl DlD2 X-ray

50

60

70

80

90

LYS LEU ASN ASP ARG ALA ASP SER ARG ARG SER LEU TRP ASP GLN GLY ASN PHE PRO LEU ILE ILE LYS ASN LEU LYS ILE GLU ASP SER ASP THR TYR ILE CYS GLU VAL GLU ASP GLN LYS GLU GLU VAL GLN LEU LEU VAL

aL

SL

CyL

“L

QL

CL

k

7L

(YL

QL

QL

(YL

QL

6L

QD

EL

7L

EL

7L

7L

YL

(YL

7L

7L

CL

(YL

aL

&L

7L

PL

CL

EL

PL

EL

7L

7L

7D

(YL

EL

CL

6L

CL

7L

7L

EL

YL

CL

aL

OL

QL

ffL

aL

EL

PL

PL

7L

ffL

ffL

aL

(YL

OL

OL

6L

PL

6L

EL

7L

CL

YL

(IL

PL

EL

EL

6D

(YL

(YL

7L

PI.

CL

EL

PL

7L

7L

7L

7D

ffL

7L

7L

CL

7L

PL

PL

7L

n

CL

ffL

bL

ffL

ffL

aL

7L

CL

PL

7L

OL

(YL

QL

ffL

aL

aL

(YD

PL

SL

CL

7L

7L

7L

(YL

PL

CL

EL

6D

(XL

aL

7L

PL

EL

7L

CL

“IL

7L

7L

ED

(YL

7L

7L

CL

PL

PL

CL

CL

EL

QL

6L

CfL

“L

OL

CL

PL

PL

PL

(YL

aL

(IL

(LL

(YL

6L

aD

PL

7L

CL

7L

CL

7L

7L

(YD

CL

CL

ffL

CyL

(YL

7L

PL

7L

EL

EL

7L

EL

7L

7D

(YL

7L

7L

EL

2

7L

CL

CL

QL (YL

aL 6,

ffL ffL

(YL 6L

(YL 6,

EL CL

PL PL

PL PL

PL 7L

aL (YL

QL (YL

QL 6,

(YL (YL

OL aL

bL 6,

ffD (YD

7L PL

7L YL

EL EL

7L EL

CL EL

7L 7L

CL EL

(YD aD

EL CL

PL PL

ffL ffL

(YL aL

aL ffL

CL

PL ;fi

7L EL

CL CL

7L PL

7L 7L

7L CL

7L 7L

7D CD

ffL 6,

7L EL

EL CL

EL PL

PL EL

PL 7L

7L CL

7L 7L

7L CL

7L EL

178 Z. Szikely et al.lJ. Mol. Struct. (Theochem) 286 (1993) 165-182

“, 8

4 d-

c

=x a e-

8 LYS46ALA oi

t

8 4 i-

.r

$8 a 6’

ASN52ALA 8 ASN52ASP

t ei

t

Fig. 4. Trajectory of the dynamical simulations of the Dl domain. The distance alteration (A) of the 46C”-52C” of the four different mutants.

Asn52Asp which showed a separation of the two biological activities as the result of the salt-bridge formation.

The results of the secondary structure analysis via our linearized notation for the Lys46Ala mutant are shown in Table 5. The information content of a single row (e.g. Ala46 corresponding to Ala46 in Lys46Ala mutant), reported in Table 5, is identical in essence to the 4,+ torsion angle

pair trajectory analysis. A thorough backbone torsion angle analysis of the Ala46 reveals that values of 4 fluctuated over the time within the range -6O”--150” while the values of @ fluctuated between +150” and 60” in the first 10 ps. In the subsequent 30 ps (10 ps < t < 40 ps) $ had settled down at +60” while C$ continued to oscillate between -60” and -150” (i.e. S, c) yL). More than 97.3% of the global conformation of 41-44,47,48

Table 4 Biological activity and variation of the 46C”-52C” distance (A) of the CD4 mutants compared to the wild-type (WT) mutant. The MHC II activities were determined by conformation dependent monoclonal antibodies (A) [11,19] and “rosette formation”

(B) 1171

Mutants of CD4 Biological activity

MHC II activity HIV gpl20 binding

Variation of 46C”-52C” distance

A B

WT full LYS46ALA reduced ASN52ALA full ASN52ASP full

100% 0%

100% no data

100% 5-7 5% 5-11

110% 5-7 24% 5-7

Tab

le

5 T

he c

onfo

rmat

iona

l al

tera

tion

of t

he a

min

o ac

id

sequ

ence

41

-60

of t

he D

l do

mai

n of

the

CD

4 m

utan

t (L

ys46

Ala

) du

ring

M

D

sim

ulat

ion.

C

onfo

rmat

ions

ar

e fr

om

a pe

riod

ical

sa

mpl

ing

usin

g a

time

inte

rval

of

2.5

ps

from

0

to 5

0 ps

. B

ackb

one

tors

ion

angl

e va

lues

fo

r a

conf

orm

atio

n ar

e re

port

ed

in T

able

1

No.

A

A

Tim

e

0 2.

5 5.

0 1.

5 10

.0

12.5

15

.0

17.5

20

.0

22.5

25

.0

27.5

30

.0

32.5

35

.0

37.5

40

.0

42.5

45

.0

47.5

50

.0

GLY

7D

^l

o 7D

7D

7D

SER

oL

aL

SL

(Y

e 6L

PHE

C

L

CL

PL

PL

PL

LH

J

EL

CL

YL

CL

EL

45

TH

R

cL

eL

eL

7~

A

ALA A

YL

6~

6~

6D

GL

Y

e,,

ro

eo

eo

to

PRO

oL

eL

eL

eL

eL

SER

CL

Ic,

PL

PL

PL

50

LY

S aL

(Y

L

QIL

(Y

L

CX

L

LE

D

6,

6~

7~

7~

6~

ASN

aL

cx

L

oL

cxL

aL

A

SP

QL

(YL

OL

(YL

AR

G

6:

7~

7~

(YL

a~

55

AL

A

eL

eL

6,_

eL

an

ASP

PL

PL

CL

PL

PL

SE

R

PL

PL

PL

PL

PL

AR

G

7~

7~

PL

7~

7~

AR

G

0~

(YL

(YL

~Y

L

7D

60

SER

a~

eo

eo

ro

e,

,

7D

6L

PL

7L

CL

6L

ED

CL

PL

(YL

aL

7D

ffL

=L

2 PL

7L

OL

CD

7D

6L

PL

7L

EL

SL

CD

EL

PL

(YL

aL

7D

(YL

aL

2 PL

YL

ffL

ED

7D

7D

3b

2;

6,

6L

7L

7L

7L

7L

7L

7L

7L

7L

7L

ED

E

D

CD

2;

EL

6,

aL

sD

SD

(YL

OL

(YL

7D

7D

7D

(YL

(YL

(YL

(YL

(YL

aL

itit

2

PL

PL

A

7L

7L

7L

(YL

OL

ffL

CD

E

D

ED

7D

ii 7L

CL

SL

CD

CL

SD

6,

‘YL

7D

ffL

aL

EL

PL

PL

7L

ffL

ED

7D

6L

PL

7L

7L

7L

ED

CL

SD

6D

(YL

7D

Q,L

aL

EL

PL A

7L

ffL

ED

7D

z 6L

PL

PL

7L

7L

7L

EL

7L

6L

ED

C

D

EL

CL

sD

sD

SD

S

D

OL

CyL

7D

7D

ffL

ffL

ffL

aL

;Jr

2

PL

A

7L

7L

CyL

Q

L

ED

E

D

7D

6,

PL

7L

7L

7L

CD

CL

6D

SD

(YL

7D

aL

(YL

EL

PL

PL

7L

(YL

CD

7D

6,

7L

7L

7L

7L

ED

CL

6D

(YL

(YL

7D

(YL

CfL

CL

PL

PL

7L

aL

ED

7D

7D

it

FL

7L

7L

7L

YL

7L

7L

CD

7D

CL

CL

PL

rc,

CyL

O

L

(YL

(YL

7D

E

D

aL

aL

(YL

(YL

2

FL

PL

A

7L

7L

(YL

(YL

CD

eD

7D

6,

SL

7L

7L

7L

CD

EL

PL

ffL

(YL

7D

CyL

aL

2 BL

7L

ffL

ED

7D

7D

it

it

7L

7L

7L

EL

7L

6L

CD

C

D

;;r

FL

(YL

QL

QL

ffL

7D

7D

OL

OL

(YL

OIL

EL

PL

2

A

PL

7L

7L

ffL

(YL

ED

E

D

Tab

le

6 T

he c

onfo

rmat

ion

alte

ratio

n of

the

am

ino

acid

seq

uenc

e 41

-60

of t

he D

l do

mai

n of

CD

4 m

utan

t (A

snSZ

Asp

) sw

ing

MD

si

mul

atio

n.

Con

form

atio

ns

are

from

a

peri

odic

al

sam

plin

g us

ing

a tim

e in

terv

al

of 2

.5 p

s fr

om

0 to

50~

s.

Bac

kbon

e to

rsio

n an

gle

valu

es

for

a co

nfor

mat

ion

is r

epor

ted

in T

able

1

No.

AA

T

ime

0.0

2.5

5.0

7.5

10.0

12

.5

15.0

17

.5

20.0

22

.5

25.0

27

.5

30.0

32

.5

35.0

37

.5

40.0

42

.5

45.0

47

.5

50.0

GL

Y

SER

PH

E

LE

U

45

TH

R

LY

S G

LY

PR

O

SER

50

L

YS

LE

U

ASP

A

SP

AR

G

55

AL

A

ASP

SE

R

AR

G

AR

G

60

SER

m

aL

EL

EL

7L

CL

CD

EL

PL

QL

6,

(YL

ffL

(YL

7L

PL

PL

7L

m

aL

mm

m

(Y

L m

m

7L

7L

7L

EL

CL

EL

C

L ;$

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Fig. 5. The distance alteration of the 46N’-52CY distance resulting from the trajectory of the MD simulation of the Dl domain of the CD4. Dl domain of the CD4 of the wild-type (upper) and Asn52Asp mutant (lower) showing the existence of the (-NH: . . .- OOC-) salt-bridge.

and 5 l-60 residues were preserved during the overall MD simulation period (12.5-50~s). However, residues 45, 46, 49 and 50 showed strong fluctua- tions. In accordance with structural expectations, as attached to biological activities, the increased motions of position 46 confirm the validity of the approach.

While portion 46, in the Lys46Ala mutant, has also shown a great deal of mobility positions 46 and 52 (the points on the salt-bridge) in the Asn52Asp mutant showed a remarkable conformational stability (Table 6). Several other amino acids (such as 41, 49, 50, 53, 54, 55, 56, 60) seemed to preserve their conformational integrity during the 12.5 ps to 50 ps MD simulation period.

Conclusions

Our main aim was to analyse the gp120 binding region of CD4 using crystallographic and computational information of various mutants. The

181

theoretical representation of the relationship between the conformational and biological binding behaviours of the CD4 mutants was success- fully performed. The main question is to understand the molecular consequences of the promising Asn52Asp mutant, in order to help us to find a minimal size region on the surface of the Dl domain showing a strong binding capacity toward gp 120.

The results obtained suggested two promising approaches of AIDS chemotherapy. One of these involve the design of cyclopepdide analogues which may cause competitive inhibition of the CD4 binding to the HIV envelope glycoprotein gp120. The second approach involves the possibility of search- ing for small molecules, which covers the gp120 binding site on the surface of CD4 without redu- cing the MHC II interaction. These potential drugs are not expected to be compounds that might cause serious conformational changes, but compounds that are capable of creating a similar electrostatic effect as observed in the case of the structure of the Asn52Asp mutant.

Acknowledgements

The authors are grateful to S. Tomlinson, (Bio- sym Inc., Munich, Germany) for providing a copy of the Biosym softwares for a short period. We thank I.G. Csizmadia (University of Toronto, Canada) and A. Aszalos (Food and Drug Administration, Washington, DC, USA) for many helpful discussions. This research was supported in part by grants from the Hungarian Scientific Foundation (OTKA No. 111-225, OTKA 616, OTKA 2703).

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A new method for the interpretation of dynamics trajectories in the conformational analysis of HIV receptor mutants