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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 calcula- tions 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 dif- ferent 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-func- tional 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 test- ing 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 require- ment 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 resolu- tion 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 bind- ing theorem in the case of wild-type (top) and for its Asn52Asp mutant (lower part). Arrows point from the pro- ton 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 simi- larity 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 recep- tor 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 muta- tion 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 replace- ment of amino acid took place at strategic posi- tions. 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 impor- tant. 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 pre- served [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 remark- able experimental result (Scheme l), a working hypothesis was established as schematically shown in Fig. 2, which provides a simple explana- tion 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 struc- ture 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 pro- teins 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 frag- ment 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 rea- son, 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 confor- mations 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 deter- mined structures was reported previously. The application of these located minima as “confor- mational 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 optimiza- tion 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 per- formed 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 calcula- tions. 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 coor- dinates 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 cen- tres” as described elsewhere [20-221. The codes and the locations of the nine “conformational centres” are summarized in Table 1. The amino acid con- formation 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 indepen- dent 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 frag- ment 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 struc- ture 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 labora- tories 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 con- formational 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 differ- ences, 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 resi- dues 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 mini- mized Dl structures (75.8%). The resemblance between the high resolution (2.3 A) X-ray struc- tures 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 struc- ture 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 ^IL 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 ^Ir.
-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 ^IL 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 ^Ir. 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 ^I~ 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)
No. AA 2cd4 Resolution: 2.4 A
4 *
lcd4 lcd4-2cd4 Resolution: 2.3 A
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 ^ID 41.91 78.75 -90.33 ^ID 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 ^I~ 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)
No. AA 2cd4 Resolution: 2.4 A
4 li,
lcd4 lcd4-2cd4 Resolution: 2.3 A
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 pre- served 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 conforma- tional 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 moni- tored 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 resi- dues 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 back- bone torsion angle analysis of the Ala46 reveals that values of 4 fluctuated over the time within the range -6O”--150” while the values of @ fluc- tuated 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
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2. Szikely et al.lJ. Mol. Struct. (Theochem) 286 (1993) M-182
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 over- all 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 conforma- tional 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 com- putational information of various mutants. The
181
theoretical representation of the relationship between the conformational and biological bind- ing 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 bind- ing 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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