Computer-assisted anti-AIDS drug development: cyclophilin B against the HIV-1 subtype A V3 loop

doi:10.4236/health.2010.27100

Paper Menu >>

Journal Menu >>

Vol.2, No.7, 661-671 (2010)

doi:10.4236/health.2010.27100

Health

Computer-assisted anti-AIDS drug development:

cyclophilin B against the HIV-1 subtype A V3 loop

Alexander M. Andrianov1*, Ivan V. Anishchenko2

1Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus;

*Corresponding Author: andrianov@iboch.bas-net.by

2United Institute of Informatics Problems, National Academy of Sciences of Belarus, Minsk, Belarus

Received 16 December 2009; revised 24 March 2010; accepted 26 March 2010.

ABSTRACT

Aim: The objects of this study originated from

the experimental observations, whereby the HIV

-1 gp120 V3 loop is a high-affinity ligand for

immunophilins, and consisted in generating the

structural complex of cyclophilin (Cyc) B be-

longing to immunophilins family with the virus

subtype A V3 loop (SA-V3 loop) as well as in

specifying the Cyc B segment forming the

binding site for V3 synthetic copy of which, on

the assumption of keeping the 3D peptide struc -

ture in the free state, may present a forward-

looking basic structure for anti-AIDS drug de-

velopment. Methods: To reach the objects of

view , molec ular docking of the HIV-1 SA-V3 loop

structure determined previously with the X-ray

conformation of Cyc B was put into practice by

Hex 4.5 program (http://www.loria.fr/~ritchied/

hex/) and the immunophilin stretch responsible

for binding to V3 (Cyc B peptide) was identified

followed by examination of its 3D structure and

dynamic behavior in the unbound status. To

design the Cyc B peptide, the X-ray conforma-

tion for the identical site of the native protein

was involved in the calculations as a starting

model to find its best energy structural variant.

The search for this most preferable structure

was carried out by consecutive use of the mo-

lecular mechanics and simulated annealing

methods. The molecular dynamics computa-

tions were implemented for the Cyc B peptide

by the GROMACS computer package (http://

www.gromacs.org/). Results: The overmolecular

structure of Cyc B with V3 was built by com-

puter modeling tools and the immunophilin-

derived peptide able to mask effectively the

structurally invariant V3 segments embracing

the functionally crucial amino acids of the HIV-1

gp120 envelope protein was constructed and

analyzed. Conclusions: Starting from the joint

analysis of the results derived w ith those of the

literature, the generated peptide was suggested

to offer a promising basic structure for making a

reality of the protein engineering projects aimed

at developing the anti-AIDS drugs able to stop

the HIV’s spread.

Keywords: HIV-1; V3 Loop; Cyclophilin B;

Computer Modeling; Molecular Docking;

Anti-Aids Dru g Design

1. INTRODUCTION

The HIV-1 envelope glycoprotein (Env), the etiologic

agent of AIDS [1], consists of two noncovalently bound

subunits derived from the gp160 precursor. One of these

subunits, gp120 protein, is localized on the surface of the

viral isolates and becomes a direct party to the virus

binding to the target-cells, whereas the other, trans-

membrane gp41 protein, triggers the process of mem-

brane fusion resulting in the invasion of the virus ge-

nome into the macrophages and T-lymphocytes [2]. Spe-

cific interactions of the HIV-1 with the virus primary

receptor CD4 as well as with its chemokine co-receptors

CCR5 and/or CXCR4 are put into effect using the

V1-V5 loops of gp120 disclosing the high variability of

the amino acid sequences in diverse virus strains [3-5].

Currently, special emphasis of the research teams in-

volved in the anti-AIDS drug studies is attracted to the

HIV-1 V3 loop (reviewed in [6]). The higher interest in

V3 is caused by numerous experimental data [7] testify-

ing to the fact that exactly this gp120 site gives rise to

the principal target for neutralizing antibodies and ac-

counts for the choice of co-receptor determining the

preference of the virus in respect with T-lymphocytes or

primary macrophages [8]. The differential usage of co-

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

662

receptors, which is critically dependent on the sequence,

charge, and/or structure of the V3 region of gp120 [9,10],

dictates the viral phenotype, which shows a typical pat-

tern of evolution during the natural history of HIV-1

infection. CCR5-restricted strains (R5) are the most

prevalent in vivo, as they are almost invariably response-

ble for the initial transmission, predominate during the

long asymptomatic phase of the infection, and often per-

sist after the progression to full-blown AIDS; by contrast,

strains that utilize CXCR4, either alone (X4) or in com-

bination with CCR5 (R5X4), emerge only in a subset of

patients, typically in conjunction with the onset of clini-

cal signs of disease progression and immune system

deterioration [11,12].

Since the V3 loop governs the cell tropism and cell

fusion [7], one of the strategic ways in developing the

anti-HIV-1 drugs may be based on the approach antici-

pating the search for the chemicals able to block effi-

ciently this functionally significant stretch of gp120 [6].

Comprehensive analysis of the data given in study [13]

allows one to suppose that immunophilins exhibiting

specific high-affinity interactions with the HIV-1 V3

loop may be utilized as a basic substance to set out of the

search for the potential anti-AIDS therapeutic agents.

Immunophilins known originally as the cellular re-

ceptors of the immunosuppressive drugs cyclosporine A

and FK506 or rapamycin organize the extensive group of

proteins exhibiting peptidyl-prolyl cis-trans isomerase

activity which is inhibited specifically and efficiently on

binding of the corresponding immunosuppressant [14].

Immunophilins subdivided into three families of proteins,

namely cyclophilins and FK506-binding proteins (FK

BPS), and a novel chimeric dual-family immunophilin,

named FK506- and Cyclosporine-binding protein

(FCBP) show similar enzymatic and biological functions

despite the apparent difference in their sequence and

three-dimensional structures [15]. Alongside with the

function of intracellular receptors of immunosuppres-

sants, individual representatives of immunophilins act as

catalysts of protein folding and as shaperones stabilizing

proteins in a defined conformation and supervising the

quality of their spatial structure [16,17]. A variety of

bacterial and protozoan pathogens express FKBP-related

peptidyl- prolyl cis-trans isomerases termed macro-

phage-infectivity potentiators (Mip). Mip proteins act in

host cell infection as virulence factors, either as mem-

brane-bound proteins on the surface of the pathogens or

as soluble secreted proteins [18,19]. The peptidyl-prolyl

cys-trans isomerase activity of Mip proteins is sup-

pressed by FK506, which reduces the infectivity of the

pathogens without affecting the rate of intracellular rep-

lication. Distinct immunophilins were found to be re-

leased from cells. Cyclophilin B was detected in human

milk [20] and blood plasma [21], but is mainly localized

in the endoplasmatic reticulum of cells. The cytosolic

immunophilins cyclophin A and FKBP12 were shown to

be released during apoptosis of fibroblasts [22] and to

act as chemokines by unknown mechanism [23-25]. Re-

cent researches have revealed that many immunophilins

possess a shaperone function independent of pepti-

dyl-prolyl cis-trans isomerase activity (reviewed in [15]).

Knockout animal studies have confirmed multiple essen-

tial roles of immunophilins in physiology and develop-

ment consisting in interactions with proteins to guide

their proper folding and assembly [15].

Reasoning from the empirical observations, there is a

good motive to think that immunophilins present in

normal human blood plasma are directly relevant to the

HIV-1 replication assisting the virus with getting into

macrophages and T-lymphocytes [26]. In particular, cy-

clophilin A packaged into nascent virus particles by spe-

cific binding to the capsid region of the Gag precursor

protein at the time of viral assembly [27-29], was found

to mediate the HIV-1 attachment to the target cells via

heparans followed by the gp120-CD4 interaction [26].

Due to the interaction of immunophilins with the HIV-1

isolates, their role of conformases or docking mediators

in the virus life cycle seems to be highly probable, since

immunophilin receptors on cell membranes and im-

munophilin-related virulence factors of pathogens have

been identified [13].

This work proceeds with our previous studies [30,31]

where two virtual molecules, namely FKBP and Cyc A

peptides, presenting the promising anti-HIV-1 pharma-

cological substances were designed by means of com-

puter modeling based on the analysis of specific interac-

tions of the FK506-binding protein and cyclophilin A

with V3.

The object of the present study was to model the

structural complex of one more protein from immuno-

philins superfamily, cyclophilin (Cyc) B, with the HIV-1

subtype A V3 loop (SA-V3 loop) circulating in Eastern

Europe including Republic of Belarus and, therefore,

offering the target of our special interest, as well as to

specify the Cyc B segment forming the binding site for

V3, the synthetic copy of which, on the assumption of

keeping the 3D peptide structure in the free state, may be

considered as a forward-looking applicant for the role of

a new antiviral drug.

To this effect, molecular docking of the HIV-1 SA-V3

structure determined previously [32] with the X-ray

conformation of Cyc B was put into practice, and the

Cyc B stretch responsible for the binding to V3 was

identified followed by predicting the most probable 3D

structure of this stretch in the unbound state, studying its

dynamic behavior, and collating the results obtained

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

663

with the X-ray data for the corresponding site of Cyc B.

Thereupon, the potential energy function was analyzed

for the complex of the SA-V3 loop with the Cyc B pep-

tide offering the virtual molecule that imitates the Cyc B

segment making a key contribution to the interactions of

the native protein with V3. As a matter of record, the

designed peptide was shown to be capable of the effect-

tive masking of the functionally critical and structurally

rigid V3 sites, presenting the suitable framework for

making a reality of the protein engineering projects util-

izing the V3 target for developing the anti-AIDS drugs

able to stop the HIV’s spread.

2. METHODS

2.1. Molecular Docking Simulations

Molecular docking of the SA-V3 loop [32] with Cyc B

(file 1CYN of the Protein Data Bank [33,34]) as well as

with the Cyc B peptide was executed by the Hex 4.5

program [35] which presents an interactive molecular

graphics package for calculating and displaying feasible

docking modes of pairs of protein and DNA molecules

and employs the spherical polar Fourier correlations to

accelerate the computations. Energy refinement of the

generated complexes was performed in the GROMACS

package [36] by minimizing their potential energy. To

this end, the conjugate gradient method was used for the

complex of the native protein with the V3 loop as well

as for the overmolecular ensemble of V3 with the Cyc B

peptide. At the final point of computations, the structural

complexes were subjected to the procedure of simulating

annealing carried out during 100 ps time domain at ini-

tial and final temperatures equal to 500 and 0 K respect-

tively.

2.2. Determination of the 3D Static Structure

for the Cyc B Peptide and Molecular

Dynamics Computations

To design the 3D structure of the Cyc B peptide, the

X-ray conformation of the Cyc B site [37] responsible

for its binding to V3 was involved in the calculations as

a starting model to find its best energy structural variant

in the unbound form. The search for this most preferable

conformation was executed by consecutive use of the

molecular mechanics and simulated annealing methods

realized in the Tinker package [38] with activating its

program modules Minimize and Anneal.

The molecular dynamics (MD) simulations of the

built Cyc B peptide structure were implemented by the

GROMACS computer package [36] using the GRO-

MOS96 force field parameter set 53A6 [39]. The starting

3D structure of the CycB peptide generated hereinbefore

was placed in a cubic box so that the smallest distance

between its walls and the peptide atoms was greater than

the half of the cut-off radius of the Coulomb and Len-

nard-Jones potentials fixed at 1.4 nm. Simple point

charge water model [40] was utilized to set the parame-

ters of explicit solvent on which the periodic boundary

conditions were imposed in all directions. Before the

MD computations, the initial Cyc B peptide model was

subjected to the procedure of energy minimization real-

ized in vacuum by the steepest descent method. The MD

simulations were carried out at temperature 310 K dur-

ing 20.5 ns time domain with 1 fs step at fixed pressure

and number of atoms, the first 0.5 ns being the stage of

solvent relaxation. To integrate the Newton’s equations

of motion, the common leap-frog algorithm was used. To

control the temperature, the weak coupling scheme to an

external bath [41] was employed in the calculations with

0.1 ps characteristic time. As with the temperature cou-

pling, the system was linked to a “pressure bath” by ex-

ponential relaxation of pressure [41] with 1.0 ps time

constant.

Every 10 ps, the geometric parameters of the MD

structures and the data on their energy characteristics

were recorded into the trajectory file. Comparison of the

MD conformations between themselves and with the

input structure was performed in terms of the values of

root-mean-square deviations computed both in Cartesian

and angular space. To this effect, the GROMACS rou-

tines [36] were implicated in the studies.

The computations were run in parallel on SKIF K-1000

computer cluster on 64 CPUs [42].

2.3. Identification of Secondary Structures

in the Cyc B Peptide

To determine the different types of secondary structures

in the Cyc B peptide, the , ψ values for all of the

amino acids derived from the simulated model were

analyzed in compliance with the criteria given in study

[43]. The types of β- and -turns were identified within

the classification of Hutchinson and Thornton [44]. To

detect the nonstandard β-turns, the additional informa-

tion on the distances Cαi…Cαi+3 computed from the atomic

coordinates of the simulated structures was employed.



2.4. Collation of 3D Static Structures

The values of root-mean-square deviations (RMSD) in

atomic coordina tes (cRMSD) were taken to evaluate the

similarity of the structures in the Cartesian space [45].

To compare the structures in terms of the dihedrals, the

RMSD between corresponding angles (aRMSD) were

used as a measure of their conformational similarity in the

angular space [45].

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

664

3. RESULTS AND DIS CUSSION Gly-17, Gln-18, Ala-19, Thr-23, and Arg-31 take up po-

sitions nearby the surface of Cyc B giving rise to the

binding site for V3 by means of Gly-1, Pro-2, Lys-3,

Gly-28, Lys-29, Thr-30, Lys-91, Lys-93, and Glu-178.

One needs to note that cooperation of the V3 loop with

Cyc B results in the origin of one ion pair organized by

Arg-31 of V3 and Glu-178 of Cyc B and in the formation

of six H-bonds that appear as a result of donor-acceptor

interactions of the receptor amino acids Lys-91, Lys-93,

and Glu-178 on the one hand as well as of V3 residues

Ser-11, Val-12, Gln-18, Ala-19, and Thr-23 on the other

hand (see information given in Table 1).

Figure 1 casts light on the structural complex of the

HIV-1 SA-V3 loop with Cyc B generated via molecular

docking of their 3D structures followed by optimization

of its geometric parameters. Insight into the function

describing the energy surface of the built complex makes

it clear that the binding of V3 to Cyc B initiates the for-

mation of stable overmolecular structure that is charac-

terized by the value of potential energy equal to –6434

kcal/mol. Analysis of the matrix of interatomic contacts

coming true in the designed complex allows one to iden-

tify the amino acids of V3 and Cyc B participating in the

intermolecular interactions the total energy of which

comes to –75 kcal/mol. So, according to the data obtained,

such V3 residues as Ser-11, Val-12, Gly-15, Pro-16,

These results signify that interaction of the V3 loop

with Cyc B entails the blockade of its central region

making the immunogenic crown of gp120 [46], whereas

the residues of V3 N- and C-terminal segments (except

Figure 1. Image of the structural complex between the HIV-1 SA-V3 loop (tubes) and Cyc B (balls).

Table 1. Geometric parameters of intermolecular H-bonds for the structural complex of the HIV-1 SA-V3 loop with Cyc B.

Residue

(donor)

Group

(donor)

Residue

(acceptor)

Group

(acceptor)

Distance (Å)

Donor…Acceptor

Distance (Å)

Hydrogen…Acceptor

Lys-932 NH Gln-181 OE1 2.7 1.7

Lys-932 NZ Thr-231 OG1 2.8 1.8

Ser-111 OG Glu-1782 OE2 2.7 1.7

Gln-181 NH Lys-912 CO 2.8 1.9

Gln-181 NE2 Glu-1782 OE2 2.8 1.9

Ala-191 NH Lys-912 CO 3.0 2.0

Footnote: Superscripts 1 and 2 denote the amino acids of V3 and Cyc B respectively.

Openly accessible at

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

665

for Arg-31) relating to its stem [46] do not come in direct

contact with the receptor. The data above are in harmony

with those of study [13], whereby high affinity to im-

munophilins is typical not merely for intact V3 variable

loops but also for their peptides embracing the immuno-

genic tip of gp120. Among the segments of V3 interact-

ing effectively with Cyc B, it is essential to mark its

tripeptide Gly-15-Pro-16-Gly-17 occurring actually in

all of the deciphered amino acid sequences of the HIV-1

principal neutralizing determinant [47]. Functional role

of this invariant V3 stretch has not been completely

specified. Nevertheless, it as known that, even a single

substitution for its central residue by alanine makes an

impact both on the virus immunogenicity and infectivity

[48] testifying to important role of Pro-16 in the HIV-1

life cycle. Under the data derived, the 3D structure of the

V3 fragment of interest is practically identical to that of

Cyc B site Gly-1-Pro-2-Lys-3 which is spatially close to

it: the value of cRMSD computed for all of the atoms of

their main chains totals 0.7 Å. Resem- blance of the 3D

main chain shapes observed for the two segments of the

ligand and the receptor makes it possible to suggest that

Cyc B stretch Gly-1-Pro-2-Lys-3 gives rise to the signal

structure that is interpreted by V3 as a mirror image of

its own immunogenic crest, which, most likely, presents

the head reason involving the specificity of V3 interac-

tions with immunophilins. In this light, the findings

above confirm the validity of the assumption made in

our previous studies [30,31] where specific high-affinity

interactions of the HIV-1 V3 variable loops with im-

munophilins arising from experimental observa- tions

[13] were suggested to be stipulated by appearing in

their amino acid sequences the fragments exposing the

similar 3D structures which are constructed from -turns

of polypeptide chain (for details see works [30,31]).

In such a way, the data of molecular docking testify to

realizing the energetically favorable contacts of the HIV-1

SA-V3 loop with Cyc B resulting in the masking of

some of the key V3 amino acids of its immunogenic

crown. In this context, we could suggest of a possible

usage of immunophilins and, in particular, Cyc B as an

alternative to the V3-directed antibodies commonly used

to neutralize the HIV-1 activity. However, the evidence

of study [49] demonstrating that increase of immuno-

philins concentration in infected blood plasma does not

influence the virus infectivity conflicts with this primi-

tive conjecture. In the case of Cyc B, the probable cause

of its insufficient neutralizing activity may consist in the

fact that, as follows from our simulations, the binding of

the immunophilin to the HIV-1 V3 loop occurs via in-

teractions with the central region of V3 and does not

affect its N- and C-terminals (Figure 1) where the major

portion of the residues involved in cell tropism and cell

fusion is localized [50-52]. Therefore, to amplify the

blockade of V3 and preserve its capacity for specific

interactions with Cyc B, we have undertaken an attempt

to design as potential anti-HIV-1 drug the virtual mole-

cule named Cyc B peptide and imitating N-terminal

segment 1-30 of the native immunophilin. The choice of

Cyc B segment 1-30 for continuation of our studies is

caused by the following motive: in compliance with the

designed data, it holds tripeptide Gly-1-Pro-2-Lys-3

recognizable by the virus immunogenic crest and com-

prises significant share of the residues making the bind-

ing site for V3. Certainly, such a definition is correct

only in case that the 3D structure of this Cyc B segment

does not experience the considerable alterations in its

free state. To check whether that is true, we computed

the most preferable 3D structure of the Cyc B peptide

and compared it with the one appearing in crystal [37] in

the corresponding site of the intact protein. Analysis of

Figure 2 illustrating the image of the superposed peptide

structures gives grounds to conclude that the spatial

folds of their backbone are closely related, and this in-

ference arising from visual observation is ratified by the

value of cRMSD equal to 2.4 Å. At collating the struc-

tures given in Figure 2, it is essential to underscore that

very close agreement between them (cRMSD is 0.5 Å)

occurs in segment Gly-1-Pro-2-Lys-3 that, as stated

above, forms in the native Cyc B the conformational

epitope specifically recognizable by V3. Analogous con-

clusion to the effect that the compared structures are

alike follows from their confrontation in the conforma-

tional space (, ): the value of aRMSD calculated for

all of the peptide amino acids comes to 33.

Insight into the static model for the 3D structure of the

Cyc B peptide (Figure 3(a)) shows that essential con-

tribution to its energy stabilization belongs to the donor-

acceptor interactions that result in forming the extensive

network of hydrogen bonds appearing between amino

acids both distant and adjacent in the polypeptide chain.

The molecule generated by computer modeling tools

offers the elongated “construction” in which the spatially

close N- and C-terminal residues give rise to the long-

range H-bond by the oxygen of Gly-1 carboxyl group

and the hydrogen of hydroxyl group of Thr-30 side chain.

As follows from the dihedral values given in Table 2,

central part 10-22 of the Cyc B peptide constitutes the

-sheet the “oval isthmus” of which is composed from

consecutive -turns (Figure 3(b)), with their spatial

folds being similar to those previously [32] in the imm-

unogenic crown of the HIV-1 SA-V3 loop. In particular,

according to our simulations, tetrapeptide Ile-14-Gly-15-

Asp-16-Glu-17 of the Cyc B peptide adopts the confor-

mation of none-standard -turn IV close to that of stretch

Gly-15-Pro-16-Gly-17-Gln-18 of V3: in this case, the

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

666

Table 2. Dihedral angles for amino acids in the 3D structure of

the Cyc B peptide.

Dihedral angles (deg.)

Residue

 ψ χ1 χ2 χ3

Gly-1 — –109.7 — — —

Pro-2 –52.2 178.6 –18.1 31.0

Lys-3 –135.3 170.1 –64.6 –176.2 –69.6

Val-4 –84.6 141.8 176.6 — —

Thr-5 –135.9 –136.8 69.1 — —

Val-6 –60.6 123.4 –62.0 — —

Lys-7 –93.7 80.2 –173.1 59.0 168.0

Val-8 –112.5 160.8 –153.8 — —

Tyr-9 –78.7 170.2 78.2 –82.8 —

Phe-10 –144.3 161.7 –168.0 –101.0 —

Asp-11 –85.7 165.1 –141.6 –58.4 —

Leu-12 –147.4 101.0 –154.5 –50.8 —

Arg-13 –129.4 102.0 –45.8 –162.4 70.9

Ile-14 –80.3 88.7 –55.0 –179.9 —

Gly-15 74.1 –72.1 — — —

Asp-16 –147.8 5.8 –152.3 –61.9 —

Glu-17 –53.7 120.6 –64.8 –176.4 –30.7

Asp-18 –74.2 42.0 –157.7 –137.1 —

Val-19 –52.9 –31.1 –165.5 — —

Gly-20 118.2 –175.8 — — —

Arg-21 –80.9 55.8 –75.7 157.4 –70.7

Val-22 –53.2 129.0 179.8 — —

Ile-23 –76.6 60.7 –39.7 –59.3 —

Phe-24 –56.9 –63.3 –66.1 –75.9 —

Gly-25 77.9 171.0 — — —

Leu-26 –138.0 159.7 –66.5 95.1 —

Phe-27 –97.0 –5.3 –51.8 –83.5 —

Gly-28 61.7 –130.8 — — —

Lys-29 –133.8 143.2 176.9 63.7 169.4

Thr-30 –122.6 — –62.2 — —

Figure 2. 3D structure of the Cyc B peptide superposed with

the X-ray conformation for segment 1-30 of the entire protein.

(a)

(b)

Figure 3. (a) Three-dimensional; (b) structures of the Cyc B

peptide generated based on the X-ray conformation of site 1-30

for the intact Cyc B.

value of cRMSD estimated for the backbone atoms of the

compared structures is 1.1 Å. Resemblance in the struc-

tural organization of the central regions of V3 and the

Cyc B peptide takes also place for their longer fragments.

For instance, if we compare the 3D structure of V3

stretch 15-20 producing the overwhelming majority of

contacts with neutralizing antibodies (study [53]) with

the one of the Cyc B peptide segment 14-19, it is also

possible to observe the conformity of their spa- tial

backbone folds (the corresponding value of cRMSD

aggregates 2.0 Å). This outcome enables one to assume

that, subject to the observance of the principle of “mirror

similarity” formulated in studies [30,31], segment of the

Cyc B peptide forming the “oval isthmus” of the -sheet

and located in the native protein inside its globule may

give an additional site for specific binding to V3 alterna-

tive to stretch Gly-1-Pro-2-Lys-3 of the intact immuno-

philin.

When looking into the secondary structure of the de-

signed molecule (Figure 3(b)), one cannot but catch

sight of the peptide segment 27-30 that, as the stretches

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

667

of its central part, exposes the conformation of -turn,

which merits the principal concern in view of the data on

the 3D structure of the SA-V3 loop [32] where the

C-terminal site organizes exactly the same structural motif.

This evidence combined with that above implicates the

following conclusion: the secondary structures of V3 and

the Cyc B peptide observed in their central and C-termi-

nal portions are closely related. In addition, considering

the main chain dihedrals (Table 2) indicates that, except

-turns, the analyzed structure also forms -bends the

central residues of which are located in positions 15, 18,

and 21 (Figure 3(b)).

The results of molecular dynamics simulations im-

plemented during 20 ns time domain by applying the

static 3D structure of the Cyc B peptide as the starting

model are evidence of its relative conformational rigidity:

the average of cRMSD calculated for the structures of

the MD trajectory and starting point amounts 3.1 Å and

the system of intramolecular H-bonds serving as one of

the factors stabilizing the molecule structure undergoes

no drastic changes within the whole MD trajectory.

Nonetheless, comprehensive study on the dynamic struc-

tures of Cyc B peptide indicates that their individual rep-

resentatives differ significantly from the input structure,

which does not exclude the probability of wide-ranging

structural reorganizations of this molecule stimulated by

abrupt alterations of the environment that, for example,

may happen upon its entry into numerous intermolecular

interactions.

Conformity of the 3D structure of the Cyc B peptide

with that of the same immunophilin segment (Figure 2)

and its relative structural inflexibility following from the

data of molecular dynamics computations give ground to

believe that the molecule designed here may not only

conserve the capacity for specific interaction with V3

characteristic of the native protein [13] but also intensify

the blockade of this cryptic site of gp120. Indeed, delv-

ing into the potential energy function describing the

structural complex of the Cyc B peptide with V3 illus-

trates (Figure 4) that, as compared to the native protein,

the peptide originating from its framework exhibits

much more extensive network of contacts with the V3

segments embracing the biologically significant amino

acids of gp120. So the energy of intermolecular interact-

tions in the complex of Cyc B with V3 amounts to –75

kcal/mol and, in the case of interest, its value falls down

to –350 kcal/mol. And at the same time, stabilization of

the complex between the Cyc B peptide and V3 is

reached owing to the multiple donor-acceptor interac-

tions (see Table 3 ) as well as to the salt bridge formed

using Arg13 of V3 and Asp-18 of immunophilin-derived

peptide. When analyzing the system of H-bonds given in

Ta bl e 3, there is need to note that, from the side of V3,

contribution to its formation belongs to such biologically

meaningful residues of gp120 as Lys-10, Arg-13, Gly-17,

Gln-18, Asp-25, Asp-29, Ile-30, and Arg-31 which find

themselves to be isolated as a result of arising the over-

molecular ensemble. Among the residues of this regis-

ter, we ought to notice Asp-25 that takes an active part in

binding of the virus to the cell membrane surface [54-58]

and, along with Ser-11, accounts for the HIV-1 pheno-

type [59,60]. Constituting the complex of the Cyc B pep-

tide with V3 also entails the masking of its functionally

critical amino acids Ser-11, Ala-19, Ile-23, Gly-24, and

Gln-32 which are also utilized by the virus to set up the

cell tropism determinant [54-58]. The active center of

V3 responsible for binding to Cyc B peptide contains

Asn-6 the blockade of which may be highly effective for

the virus inactivation: as mentioned above, this amino

acid of V3 presenting the integral part of its structurally

invariant segment 3-7 [32] gives rise to one of the con-

served sites of N-linked glycosylation of gp120 [61].

When examining the overmolecular ensemble repre-

sented in Figure 4, one needs to cast a glance at the fol-

lowing feature: in this complex, segment Gly-1-Pro-2-

Lys-3 of the Cyc B peptide interacts with structurally

rigid stretch 28-32 of the HIV-1 V3 domain [32], whereas

the corresponding site of the native protein, as stated

before, contacts the immunogenic crown of gp120 (Fig-

ure 1). At the same time, this V3 region that proves to be

unused by the N-terminal site of the Cyc B peptide be-

comes very intimate with pentapeptide Ile-14-Gly-15-

Asp-16-Glu-17-Asp-18 belonging to the “oval isthmus”

of its -sheet. These observations are of special interest

since the indicated segments of the receptor and ligand

reside in the -turns of polypeptide chain which may

serve as docking sites for protein-protein interactions

[62-64]. The presence of -turns in the 3D structures of

V3 and the Cyc B peptide is likely to be one of the head

factors that may make a determinative contribution to

the specificity of their efficacious interactions.

Collating the 3D structures of V3 and the Cyc B pep-

tide in natural and constrained states indicates that, in

either case, forming the complex brings in the certain

structural rearrangements taking place both in the Carte-

sian and angular spaces. At the same time, the Cyc B

peptide experiences the more profound transformation of

its structure: so when we confront the V3 structures ma-

terialized in the overmolecular ensemble and in the un-

bound status, the values of cRMSD and aRMSD are

respectively 2.0 Å and 47 and, in the case of the Cyc B

peptide, the corresponding values rise to 4.0 Å and 57.

This observation falls into line with the supposition

above, whereby the Cyc B peptide, in spite of the rela-

tive conformational rigidity, may exhibit the higher

flexibility of the polypeptide chain on drastic medium

alterations.

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

668

Figure 4. Overmolecular structure of the HIV-1 SA-V3 loop (balls) with the Cyc B peptide (tubes).

Table 3 . Geometric parameters of intermolecular H-bonds for the structural complex of the HIV-1 SA-V3 loop with the Cyc B

peptide.

Residue

(donor)

Group

(donor)

Residue

(acceptor)

Group

(acceptor)

Distance (Å)

Donor…Acceptor

Distance (Å)

Hydrogen…Acceptor

Arg-131 NH1 Asp-182 OD1 3.0 2.0

Arg-131 NH2 Asp-182 OD1 3.2 2.3

Arg-131 NH2 Asp-182 OD2 3.1 2.2

Gly-171 NH Asp112 OD1 2.9 1.9

Gln-181 NE2 Tyr-92 CO 2.8 1.8

Gln-181 NE2 Asp-112 OD1 2.8 1.8

Asp-251 NH Gly-282 CO 2.8 1.8

Lys-32 NZ Ile-301 CO 2.8 1.9

Thr-52 OG1 Arg-311 CO 2.7 1.7

Lys-72 NH Lys-101 CO 2.8 1.8

Lys-292 NZ Asp-291 CO 2.9 2.0

Footnote: Superscripts 1 and 2 denote the amino acids of V3 and the CycB peptide respectively.

4. CONCLUSIONS

In studies [30,31], we implemented the computer-aided

design of two molecules referred to as Cyc A and FKBP

peptides and, having analyzed their structural complexes

with the HIV-1 SA-V3 loop [32], disclosed that the Cyc

A peptide binds effectively to its immunogenic crown,

whereas the FKBP peptide prefers to interact with the N-

and C-terminal segments of the virus principal neutral-

izing determinant. The findings derived here bear wit-

ness that, unlike the molecules constructed previously

[30,31], the Cyc B peptide is able to mask the function-

Openly accessible at

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

669

ally crucial amino acids both of the V3 central part and

of its stem stretches. Moreover, as compared to these

molecules, cooperation of the Cyc B peptide with V3

brings in the origin of more stable overmolecular struc-

ture: for instance, the value of the energy of intermo-

lecular interactions computed for the structural complex

of V3 with Cyc A peptide [31] totals -87 kcal/mol and, in

the case in question, it aggregates -350 kcal/mol (see

above).

As shown in study [32], in spite of the hypervariabil-

ity of V3, its segments 3-7, 15-20, and 28-32 embracing

the highly conserved amino acids of gp120 give rise to

the closely related spatial backbone folds in different

virus isolates, and, therefore, they may be considered as

promising targets for anti-AIDS drug studies. Allowing

for these data in common with the evidence which bears

witness that the Cyc B peptide is capable of masking the

V3 functionally critical residues residing in its structur-

ally invariant segments, one may expect that synthetic

copy of this virtual molecule (or its structural analogs)

may display biological activity to various HIV-1 strains

exhibiting a broadly neutralizing effect. Beyond all

shadow of doubt, the peptide constructed here must ex-

perience the extensive experimental test to be considered

as the coming applicant for the role of “magic bullet”

displaying a wide-range blockade of the HIV-1 envelope

glycoprotein gp120.

In conclusion, the model of the structural complex of

Cyc B with V3 proposed above substantiates the litera-

ture data on a high affinity of immunophilins to V3 [13],

and the results derived from its analysis enable one to

make an optimistic prognosis of the prospects of using

their peptides as the starting chemicals for the design of

efficacious antiviral agents by protein engineering me-

thods.

5. ACKNOWLEDGEMENTS

This study was supported by grants from the Union State of Russia and

Belarus (scientific program SKIF-GRID; № 4U-S/07-111) as well as

from the Belarusian Foundation for Fundamental Research (project

X08-003).

REFERENCES

[1] Gallo, R.C. and Montagnier, L. (2003) The discovery of

HIV as the cause of AIDS. The New England Journal of

Medicine, 349(24), 2283-2285.

[2] Wyatt, R. and Sodroski, J. (1998) The HIV-1 envelope

glycoproteins: Fusogens, antigens, and immunogens.

Science, 280(5371), 1884-1888.

[3] Landau, N.R., Warton, M. and Littman, D.R. (1988) The

envelope glycoprotein of the human immunodeficiency

virus binds to the immunoglobulin-like domain of CD4.

Nature, 334(6178), 159-162.

[4] Feng, Y., Broder, C.C., Kennedy, P.E. and Berger, E.A.

(1996) HIV-1 entry cofactor: Functional cDNA cloning

of a seven-transmembrane, G protein-coupled receptor.

Science, 272(5263), 872-877.

[5] Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D.,

Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R.E.,

Hill, C.M., Davis, C.B., Peiper, S.C., Schall, T.J., Littman,

D.R. and Landau, N.R. (1996) Identification of a Major

Co-Receptor for Primary Isolates of HIV-1. Nature,

381(6584), 661-666.

[6] Sirois, S., Sing, T. and Chou, K.C. (2005) HIV-1 gp120

V3 loop for structure-based drug design. Current Protein

& Peptide Science, 6(5), 413-422.

[7] Hartley, O., Klasse, P.J., Sattentau, Q.J. and Moore J.P.

(2005) V3: HIVs Switch-Hitter. AIDS Research and

Human Retroviruses, 21(8), 171-189.

[8] Hwang, S.S., Boyle, T.J., Lyerly, H.K. and Cullen, B.R.

(1991) Identification of the envelope V3 loop as the pri-

mary determinant of cell tropism in HIV-1. Science,

253(5015), 71-74.

[9] Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B.,

Ponath, P.D., Wu, L., Mackay, C.R., LaRosa, G., New-

man, W., Gerard, N., Gerard, C. and Sodroski, J. (1996)

The beta-chemokine receptors CCR3 and CCR5 facilitate

infection by primary HIV-1 isolates. Cell, 85(7), 1135-

1148.

[10] Cocchi, F., DelVico, A., Garzino-Demo, A., Cara, A.,

Gallo, R.C. and Lusso, P. (1996) The V3 domain of the

HIV-1 gp120 envelope glycoprotein is critical for che-

mokine-mediated blockade of infection. Nature Medicine,

2(11), 1244-1247.

[11] Connor, R.I., Sheridan, K.E., Ceradini, D., Choe, S. and

Landau, N.R. (1997) Change in coreceptor use correlates

with disease progression in HIV-1-infected individuals.

The Journal of Experimental Medicine, 185(4), 621-628.

[12] Scarlatti, G., Tresoldi, E., Bjorndal, A., Fredriksson, R.,

Colognesi, C., Deng, H.K., Malnati, M.S., Plebani, A.,

Siccardi, A.G., Littman, D.R., Fenyo, E.M. and Lusso, P.

(1997) In vivo evolution of HIV-1 co-receptor usage and

sensitivity to chemokine-mediated suppression. Nature

Medicine, 3(11), 1259-1265.

[13] Endrich, M.M. and Gehring, H. (1998) The V3 loop of

human immunodeficiency virus type-1 envelope protein

is a high-affinity ligand for immunophilins present in

human blood. European Journal of Biochemistry, 252(3),

441-446.

[14] Galat, A. and Metcalfe, S.M. (1995) Peptidylproline

cis/trans isomerases. Progress in Biophysics and Mo-

lecular Biology, 63(1), 67-118.

[15] Barik, S. (2006) Immunophilins: For the love of proteins.

Cellular and Molecular Life Sciences, 63(24), 2889-

2900.

[16] Baker, E.K., Colley, N.J. and Zuker, C.S. (1994) The

cyclophilin homolog NinaA functions as a chaperone,

forming a stable complex in vivo with its protein target

rhodopsin. The EMBO Journal, 13(20), 4886-4895.

[17] Ferreira, P.A., Nakayama, T.A. and Travis, G.H. (1997)

Interconversion of red opsin isoforms by the cyclophilin-

related chaperone protein Ran-binding protein 2. Pro-

ceedings of the National Academy of Sciences, USA,

94(4), 1556-1561.

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

670

[18] Hacker, J. and Fischer, J. (1993) Immunophilins: struc-

ture-function relationship and possible role in microbial

pathogenicity. Molecular Microbiology, 10(3), 445-456.

[19] Moro, A., Ruiz-Cabello, F., Fernandez-Cano, A., Stock,

R. P. and Gonzalez, A. (1995) Secretion by Trypanosoma

cruzi of a peptidyl-prolyl cis-trans isomerase involved in

cell infection. The EMBO Journal, 14(11), 2483-2490.

[20] Spik, G., Haendler, B., Delmas, O., Mariller, C., Chamoux,

M., Maes, P., Tartar, A., Montreuil, J., Stedman, K., Ko-

cher, H.P., Keller, R., Hiestand, P.C. and Movva, N.R.

(1991) A novel secreted cyclophilin-like protein (SCYLP).

Journal of Biological Chemistry, 266(17), 10735-10738.

[21] Allain, F., Boutillon, C., Mariller, C. and Spik, G. (1995)

Selective assay for CyPA and CyPB in human blood us-

ing highly specific anti-peptide antibodies. Journal of

Immunological Methods, 178(1), 113-120.

[22] Endrich, M. M., Grossenbacher, D., Geistlich, A. and Ge-

hring, H. (1996) Apoptosis-induced concomitant release

of cytosolic proteins and factors which prevent cell death.

Biology of the Cell, 88(1-2), 15-22.

[23] Sherry, B., Yarlett, N., Strupp, A. and Cerami, A. (1992)

Identification of cyclophilin as a proinflammatory secre-

tory product of lipopolysaccharide-activated macrophages.

Proceedings of the National Academy of Sciences, USA,

89(8), 3511-3515.

[24] Xu, Q., Leiva, M.C., Fischkoff, S.A., Handschumacher,

R.E. and Lyttle, C.R. (1992) Leukocyte chemotactic ac-

tiveity of cyclophilin. The Journal of Biological Chemis-

try, 267(17), 11968-11971.

[25] Bang, M., Muller, W., Hans, M., Brune, K., Swandulla, D.

(1995) Activation of Ca2+ signaling in neutrophils by the

mast cell-released immunophilin FKBP12. Proceedings

of the National Academy of Sciences, USA, 92(8),

3435-3438.

[26] Saphire, A.C.S., Bobardt, M.D. and Gallay, P.A. (1999)

Host cyclophilin A mediates HIV-1 attachment to target

via heparans. The EMBO Journal, 18(23), 6771-6785.

[27] Franke, E.K., Yuan, H.E.H. and Luban, J. (1994) Spe-

cific incorporation of cyclophhilin a into HIV-1 virions.

Nature, 372(6504), 359-362.

[28] Thali, M., Bukovsky, A., Kondo, E., Rosenwirth, B.,

Walsh, C.T., Sodroski, J. and Gottlinger, H.G. (1994)

Functional association of cyclophilin A with HIV-1 viri-

ons. Nature, 372(6504), 363-365.

[29] Colgan, J., Yuan, H.E.H., Franke, E.K. and Luban, J.

(1996) Binding of the human immunodeficiency virus

type 1 Gag polyprotein to cyclophilin A is mediated by

the central region of capsid and requires Gag dimeriza-

tion. The Journal of Virology, 70(7), 4299-4310.

[30] Andrianov, A.M. (2008) Computational anti-AIDS drug

design based on the analysis of the specific interactions

between immunophilins and the HIV-1 gp120 V3 loop.

Application to the FK506-binding protein. Journal of

Biomolecular Structure & Dynamics, 26(1), 49-56.

[31] Andrianov, A.M. (2009) Immunophilins and HIV-1 V3

loop for structure-based anti-AIDS drug design. Journal

of Biomolecular Structure & Dynamics, 26(4), 445-454.

[32] Andrianov A.M. and Anishchenko I.V. (2009) Compu-

tational model of the HIV-1 subtype A V3 loop: Study on

the conformational mobility for structure-based anti-

AIDS drug design. Journal of Biomolecular Structure &

Dynamics, 27(2), 179-194.

[33] Bernstein, F.C., Koetzle, T.F., Williams, G.J.B., Meyer, E.

F., Brice, M.D., Rodgers, J.R., Kennard, O., Shima-

nouchi, T. and Tasumi, M. (1997) The protein data bank.

A computer-based archival file for macromolecular struc-

tures. Journal of Molecular Biology, 112(3), 535-542.

[34] Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G.,

Bhat, T.N., Weissig, H., Shindyalov, I.N. and Bourne, P.E.

(2000) The Protein Data Bank. Nucleic Acids Research,

28(1), 235-242.

[35] Mustard, D. and Ritchie, D.W. (2005) Macromolecular

docking using spherical polar fourier correlations. Pro-

teins: Structure, Function, and Bioinformatics, 60(2),

269-274.

[36] Berendsen, H.J.C., van der Spoel, D. and van Drunen, R.

(1995) GROMACS: A message-passing parallel mo-

lecular dynamics implementation. Computer Physics Co-

mmunications, 91(1-3), 43-56.

[37] Mikol, V., Kallen, J. and Walkinshaw, M.D. (1994) X-ray

structure of a Cyclophilin B/Cyclosporin complex: Com-

parison with Cyclophilin A and delineation of its cal-

cineurin-binding domain. Proceedings of the National

Academy of Sciences, 91(11), 5183-5186.

[38] Ren, P. and Ponder, J.W. (2003) TINKER: Software tools

for molecular design. The Journal of Biological Chemis-

try, 107, 5933-5947.

[39] Oostenbrink, C., Villa, A., Mark, A.E. and van Gunsteren,

W.F. (2004) A biomolecular force field based on the free

enthalpy of hydration and solvation: The GROMOS

force-field parameter sets 53A5 and 53A6. The Journal

of Biological Chemistry, 25(13), 1656-1676.

[40] Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F.

and Hermans, J. (1981) Interaction models for water in

relation to protein hydration. In: B. Pullman Ed., Inter-

molecular Forces, Dordrecht: D. Reidel Publishing Com-

pany, Boston, pp. 331-342.

[41] Berendsen, H.J.C., Postma, J.P.M., DiNola, A. and Haak,

J.R. (1984) Molecular-dynamics with coupling to an ex-

ternal bath. Journal of Chemical Physics, 81(8), 3684-

3690.

[42] Ablameyko, S.V., Abramov, S.M., Anishchanka, U.V.,

Medvedev, S.V., Paramonov, N.N. and Tchij, O.P. (2005)

SKIF supercomputer configurations (in Russian). Minsk,

United Institute of Informatics Problems.

[43] Smith, L.J., Bolin, K.A., Schwalbe, H., MacArthur, M.

W., Thornton, J.M. and Dobson, C.M. (1996) Analysis of

main chain torsion angles in proteins: Prediction of NMR

coupling constants for native and random coil conforma-

tions. Journal of Molecular Biology, 255(3), 494-506.

[44] Hutchinson, E.G. and Thornton, J.M. (1996) PROMO-

TIF—a program to identify and analyze structural motifs

in proteins. Protein Science, 5(2), 212-220.

[45] Sherman, S.A. and Johnson, M.E. (1993) Derivation of

locally accurate spatial protein structure from NMR data.

Progress in Biophysics and Molecular Biology, 59(3),

285- 339.

[46] Cormier, E.G. and Dragic, T. (2002) The crown and the

stem V3 loop play distinct roles in human immunodefi-

ciency virus type 1 envelope glycoprotein interactions

with CCR5 coreceptor. The Journal of Virology, 76(17),

8953-8957.

[47] LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A.,

Profy, A.T., Lewis, J.A., Langlois, A.J., Dressman, G.R.,

A. M. Andrianov et al. / HEALTH 2 (2010) 661-671

671

Boswell, R.N., Shadduk, P., Holley, L.H., Karplus, M.,

Bolognesi, D.P., Matthews, T.J., Emini, E.A. and Putney,

S.D. (1990) Conserved sequence and structural elements

in the HIV-1 principal neutralizing determinant. Science,

249(4971), 932-935.

[48] Ivanoff, L.A., Looney, D.J., McDanal, C., Morris, J.F.,

Wong-Staat, F., Lang, A.J., Petteway, S.R.Jr. and Mat-

thews, T.J. (1991) Alteration of HIV-1 infectivity and

neutralization by a single amino acid replacement in the

V3 loop domain. AIDS Research and Human Retrovi-

ruses, 7(7), 595-603.

[49] Minder, D., Boni, J., Schupbach, J. and Gering, H. (2002)

Immunophilins and HIV-1 infection. Archives of Virology,

147(8), 1531-1542.

[50] Wang, W.-K., Dudek, T., Zhao, Y.-J., Brumblay, H.G.,

Essex, M. and Lee, T.-H. (1998) CCR5 coreceptor utili-

zation involves a highly conserved arginine residue of

HIV type 1 gp120. Proceedings of the National Academy

of Sciences, USA, 95(10), 5740-5745.

[51] de Parseval, A., Bobardt, M.D., Chatterji, U., Elder, J.H.,

David, G., Zolla-Pazner, S., Farzan, M., Lee, T.H. and

Gallay, P.A. (2005) A highly conserved arginine in gp120

governs HIV-1 binding to both syndecans and CCR5 via

sulfated motifs. The Journal of Biological Chemistry,

280(47), 39493-39504.

[52] Hu, Q., Napier, K.B., Trent, J.O., Wang, Z., Taylor, S.,

Griffin, G.E., Peiper, S.C. and Shattock, R.J. (2005) Re-

stricted variable residues in the C-terminal segment of

HIV-1 V3 loop regulate the molecular anatomy of CCR5

utilization. Journal of Molecular Biology, 350(4), 699-

712.

[53] Ghiara, J.B., Stura, E.A., Stanfield, R.L., Profy, A.T. and

Wilson, I.A. (1994) Crystal structure of the principal

neutralization site of HIV-1. Science, 264(5155), 82-85.

[54] Chavda, S.C., Griffin, P., Han-Liu, Z., Keys, B., Vekony,

M.A. and Cann, A.J. (1994) Molecular determinants of

the V3 loop of human immunodeficiency virus type 1

glyco-protein gp120 responsible for controlling cell tro-

pism. Journal of General Virology, 75(11), 3249-3253.

[55] Mammano, F., Salvatori, F., Ometto, L., Panozzo, M.,

Chieco-Bianchi, L. and De Rossi, A. (1995) Relationship

between the V3 loop and the phenotypes of human im-

munodeficiency virus type 1 (HIV-1) isolates from chil- -

dren perinatally infected with HIV-1. The Journal of Vi-

rology, 69(1), 82-92.

[56] Milich, L., Margolin, B.H. and Swanstrom, R. (1993) v3

loop of the human immunodeficiency virus type 1 env

protein: Interpreting sequence variability. The Journal of

Virology, 67(9), 5623-5634.

[57] Shioda, T., Levy, J.A. and Cheng-Mayer, C. (1992) Small

amino acid changes in the V3 hypervariable region of

gp120 can affect the T-cell-line and macrophage tro-

pism of human immunodeficiency virus type 1. Pro-

ceedings of the National Academy of Sciences, 89(20),

9434-9438.

[58] Wu, L., Gerard, N.P., Wyatt, R., Choe, H., Parolin, C.,

Ruffin, N., Borsetti, A., Cardoso, A.A., Desjardin, E.,

Newman, Gerard, W.C. and Sodroski, J. (1996) CD4-in-

duced interaction of primary HIV-1 gp120 glycoproteins

with the chemokine receptor CCR-5. Nature, 384(6605),

179-183.

[59] Fouchier, R.A.M., Groenink, M., Kootstra, N.A., Ters-

mette, M., Huisman, H.G., Miedema, F. and Schuite-

maker, H. (1992) Phenotype-associated sequence variation

in the third variable domain of the human immunodefi-

ciency virus type 1 gp120 molecule. The Journal of Vi-

rology, 66(5), 3183-3187.

[60] De Jong, J.J., De Ronde, A., Keulen, W., Tersmette, M.

and Goudsmit, J. (1992) Minimal requirements for the

human immunodeficiency virus type 1 V3 domain to

support the syncytium-inducing phenotype: Analysis by

single amino acid substitution. The Journal of Virology,

66(11), 6777-6780.

[61] Ogert, R.A., Lee, M.K., Ross, W., Buckler-White, A.,

Martin, M.A. and Cho, M.W. (2001) N-linked glycosyla-

tion sites adjacent to and within the V1/V2 and the V3

loops of dualtropic human immunodeficiency virus type

1 isolate DH12 gp120 affect coreceptor usage and cellu-

lar tropism. The Journal of Virology, 75(13), 5998-6006.

[62] Smith, J.A. and Pease, L.J. (1980) Reverse turns in pep-

tides and proteins. Critical Reviews in Biochemistry, 8(4),

315-399.

[63] Rose, G.D., Gierasch, L.M. and Smith, J.A. (1985) Turns

in peptides and proteins. Advances in Protein Chemistry,

37, 1-109.

[64] Newton, A.C. (2001) Protein kinase C: Structural and

spatial regulation by phosphorylation, cofactors, and

macromolecular interactions. Chemical Reviews, 101(8),

2353-2364.