Báo cáo khoa học: Adaptability and flexibility of HIV-1 protease potx

To analyse further the behaviour of these catalytic aspartates, we have extended our comparison to structures of C95A mutant protease complexed with different inhibitors.. Superposition

Adaptability and flexibility of HIV-1 protease

Mukesh Kumar and Madhusoodan V Hosur

Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, India

Even though more than 200 three-dimensional structures

of HIV-1 protease complexed to a variety of inhibitors

are available in the Protein Data Bank; very few

struc-tures of unliganded protein have been determined We

have recently solved structures of unliganded HIV-1

protease tethered dimer mutants to resolutions of 1.9 A˚

and 2.1 A˚, and have found that the flaps assume

closed-flap conformation even in the absence of any bound

lig-and We report comparison of the unliganded closed-flap

structure with structures of HIV-1 protease inhibitor

complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexi-bility and adaptaflexi-bility of proteins in general

Keywords: adaptability; flexibility; HIV-1 protease; inhibi-tors; structure

The significance of human immunodeficiency virus type 1

(HIV-1) protease in the life cycle of HIV has made it a prime

therapeutic target for the development of anti-HIV drugs

This has resulted in the determination of a large number of

structures of identical or closely related sequences with

different ligands by X-ray, NMR and theoretical molecular

modelling approaches To date a total of 213 such structures

are available from the Protein Data Bank (PDB) [1] and

HIV-protease database (http://home2.ncifcrf.gov/HIVdb)

In spite of such a large number of studies on a single system

consisting of closely related sequences, we still lack the

proper understanding to tackle the problem of drug

resistance mutations, a typical characteristic associated with

HIV infection This inability to understand the behaviour of

the protease demands the development of more

sophisti-cated tools for the study of protein structure There is an

inevitable need to improve our knowledge of the inherent

flexibility and adaptability in the three-dimensional

struc-ture of proteins These two feastruc-tures of proteins are as

important as the structure itself and are very often

responsible for the functional characteristics of a particular

structure under different ‘natural’ and ‘stressed’

environ-ments As reported by Zoete et al [2], X-ray structures

through the B-factors, molecular dynamics simulations and

normal mode analyses give a fair idea about the fluctuations

of different residues/regions of proteins about their mean

positions This mobility may be described as the flexibility of

the region In contrast, adaptability of a residue is the ability

of that residue to alter its mean position itself in response to changes in its chemical environment Information about residue adaptabilities can be obtained only by detailed comparison of the protein structures in the presence and absence of the environmental stress, which may be in the form of a point mutation or a bound ligand We determined earlier the structure of the double mutant C95M/C1095A in

an unliganded state By comparison with the isomorphous structure of unliganded C95M single mutant, we analysed [3] the effect of C95A mutation on the structure of the protease itself In addition to the changes on the dimer interface, we observed that the catalytic aspartates Asp25/

1025 and the catalytic water move to make this catalytic site more accessible to the substrate (Fig 1)

This structural change in the active site was subsequently shown to be associated with increased autolysis rates in protease carrying the C95A mutation [3] To analyse further the behaviour of these catalytic aspartates, we have extended our comparison to structures of C95A mutant protease complexed with different inhibitors We find that the polypeptide chain segment 23–26, which includes the catalytic aspartates, is structurally most adaptable even though it is least flexible This observation is significant and can contribute to the fact that HIV-1 protease enzyme has the unique property of cleaving protein substrates at eight different amino acid sequences [4]

Materials and methods

‘Closed-flap’ structure of unliganded HIV-1 protease The X-ray structures of C95M single mutant- and C95M/ C1095A double mutant-tethered HIV-1 protease have been solved to 1.9 A˚ and 2.1 A˚ resolution, respectively, and deposited in the PDB(files 1G6L and 1LV1) Residues in the first monomer have been numbered 1–99 and those in the second monomer 1001–1099 Residues in the linker are numbered 101–105 Electron density was not visible for the linker peptide suggesting that the linker was highly flexible

Correspondence to M V Hosur, Solid State Physics Division,

Bhabha Atomic Research Centre, Mumbai, India-400085.

Fax: +91 22 5515050, Tel.: +91 22 5593614,

E-mail: hosur@magnum.barc.ernet.in

Abbreviations: HIV-1, human immunodeficiency virus type 1;

Rmsd, root mean square deviation.

Enzyme: HIV-1 protease (EC: 3.4.23.16).

(Received 2 August 2002, revised 14 November 2002,

accepted 27 January 2003)

Eur J Biochem 270, 1231–1239 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03483.x

These two structures revealed for the first time the

closed-flap conformation of HIV-1 protease even in the absence of

any ligand bound in the active site [3,5] Comparison of such

a structure with the closed-flap ligand-bound structure is

expected to be more rational and useful

Inhibitor complex structures containing C95A HIV-1

protease

Eight structures deposited in the PDB(files 1DAZ, 1A94,

1HWR, 1HVR, 1QBS, 1DMP, 1QBR and 1QBT) are of

complexes between different types of inhibitor molecules

and HIV-1 protease containing the C95A mutation In two

of these structures (1DAZ and 1A94), the protein is

complexed with a peptide inhibitor [6,7] and in the

remaining six (1HWR, 1HVR, 1QBS, 1DMP, 1QBR and

1QBT) the enzyme is complexed with a cyclic urea-based

inhibitor [8–13] All the cyclic urea-based inhibitors contain

a central seven-membered ring having a urea moiety, and a

diol group P1/P1¢ substituents are attached to C3/C6 atoms

of the central ring and P2/P2¢ substituents are attached to

urea nitrogen atoms of the ring as shown in Fig 2A The

seven membered ring sits in the active site cavity as a bridge

between the flaps and the catalytic aspartates While the

urea moiety makes hydrogen bonds with main chain NH of

the flap residue Ile50 from both the monomers, the diol

group makes hydrogen bonds with the two catalytic residues

Asp25 and Asp1025 The P2 moities are all different among

the six inhibitors listed in Fig 2B

All of the superposition and structure comparisons were

performed using software O [14] and molecular images

were made using MOLRAY [15] All structure alignments

were based on superposition of Ca atoms of all residues of

the entire HIV-1 protease dimer With the whole structures

so superposed, the root mean square deviations (Rmsd

values) quoted for any polypeptide segment is then

calcu-lated for the Ca atoms of the amino acid residues in that

polypeptide segment Error-scaled difference distance

mat-rix plots were generated using the program [16] The

errors in atomic positions for error-scaling were estimated using ‘diffraction-component precision index’ plus linear B-scaling [17]

Results

Structural features of C95A mutation in HIV-1 protease Fig 3 shows a B-factor vs residue number plot for C95M/ C1095A HIV-1 protease It may be seen in the figure that the residues 23–26 in the first monomer and 1023–1026 in the second monomer located in the active site of protease have the lowest B-factors suggesting that these residues are relatively ‘rigid’ entities in the protein structure The electron density in this region is also very well defined indicating that these residues have very well-defined conformation Karplus and his colleagues have drawn a similar conclusion recently, after analysing 73 crystal structures of HIV-1 protease complexed to a variety of inhibitors Comparison of the C95M/C1095A double mutant with the C95M single mutant HIV-1 protease structure by superposition of all

198 Ca atom pairs showed that the main chain of the residues 23–26/1023–1026 has moved towards the flap after C1095A mutation Interestingly, this movement includes movement of catalytic aspartates Asp25/1025 along with the bound catalytic water (Fig 1) in a direction that would enable more access to the catalytic centre for the incoming substrate Eventually this resulted in increased autolysis after C1095A mutation [3] Therefore, this subtle movement

in the catalytic centre is of prime importance from the point

of view of enzymatic activity These atomic movements were also verified by means of difference distance matrix plots, which have the advantage of not depending on any particular frame of superposition These plots are shown

in Fig 4 for one subunit In Fig 4A the upper right triangle shows the normal difference distance matrix plot while the lower left triangle shows error-scaled difference distance matrix plot for the structures 1LV1 vs 1G6L It may be seen that the region 23–26 is involved in significant atomic Fig 1 Superposition of catalytic aspartates of C95M HIV-1 protease (shown by green carbons) and C95M/C1095A HIV-1 protease (shown by yellow carbons).

movement The error-scaled difference distance matrix plot

also shows that this segment in 1LV1 has moved towards

the flaps and along the direction joining the active site

aspartates to the tips of the flaps

Structural effect of inhibitor binding on HIV-1 protease The closed-flap structure of the unliganded C1095A mutant enables, through structural comparisons, accurate Fig 2 Basic structure of P2 analogues of cyclic urea inhibitors (A) and properties and structures of all inhibitors (B).

FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur J Biochem 270) 1233

identification of the changes, if any, brought about by

inhibitor binding The unliganded and all six of the urea

inhibitor complex structures belong to the same space group

(Fig 2B) further enhancing the meaningfulness of these

comparisons Table 1 shows the Rmsd values for pair-wise superposition of the unliganded C95M/C1095A structure with each of the six cyclic urea-complexed structures listed

in Fig 2B

Fig 3 Average B-factors for different residues

of C95M/C1095A HIV-1 protease.

Fig 4 Difference distance matrix plot The upper right triangle shows the normal difference distance matrix plot and the lower left triangle shows the error-scaled difference distance matrix plot (A) 1LV1 vs 1G6L (A) 1LV1 vs 1QBS.

The corresponding Rmsd for superposition onto the

1DAZ structure, which belongs to the orthorhombic crystal

system, is 0.45 A˚ Thus, in spite of different crystal

environments, the overall conformation of HIV-1 protease

is very similar, with Rmsd values ranging from 0.26

to 0.45 A˚ However, there are subtle differences

character-istic of the bound ligand Fig 5 shows the superposition of

peptide complex structure 1DAZ with unliganded structure

1LV1 Interestingly, the catalytic aspartates Asp25/1025 in

the two structures (1LV1 and 1DAZ) are very much super

imposable, and the Rmsd for residues 23–26/1023–1026 is

0.11 A˚ Similar is the case with another peptide–inhibitor

complex (PDBcode 1A94) suggesting that the linear chain

of peptide inhibitors does not cause much alteration in the

position of catalytic aspartates

However, when the cyclic urea complex structures are

superimposed on to the unliganded structure 1LV1, the

position of the main chain of the catalytic residues

Asp25/1025 is significantly different (see Fig 7) The shift

of the Ca atoms of Asp25/1025) when averaged over

all six comparisons) is 0.37 A˚, and has apparently been induced by the inhibitor to relieve what would otherwise

be bad steric contacts (2.7 A˚ and 2.81 A˚) between the cyclic urea ring of the inhibitor and the aspartate side chains, as shown in Fig 6 This movement of catalytic Asp25 is also revealed by the error-scaled difference distance matrix plot (Fig 4B) for comparison of 1LV1 and 1QBS structures The residues 23–26 in 1QBS have moved away from the flap-tips, and to the direction joining active-site aspartates to flap-tips, in agreement with results of molecular superposition (Fig 7) It thus appears that the position of polypeptide chain segment 23–26/1023–1026 is influenced by both mutational stress C95A (Fig 1) below the active site cavity and also by steric stress from inhibitor binding in the active site cavity (Fig 7) Thus, of utmost interest is the fact that what is considered the most rigid polypeptide segment in the active site of HIV-1 protease can alter its position when required Structural rearrangement in the form of movements of the side-chain atoms in the S1 pocket on

Table 1 Rmsd values for pair-wise superposition of unliganded C95M/C1095A structure witheachof the six cyclic urea-complexed structures shown in Fig 2B.

Fig 5 Superposition of peptide inhibitor-bound C95A HIV-1 protease in PDB file 1DAZ (shown by green carbons) on unliganded HIV-1 protease structure in PDB file 1LV1 (shown by yellow carbons) The water molecule is present only in the unliganded structure.

FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur J Biochem 270) 1235

inhibitor binding has been recently observed

crystallo-graphically [18] However, the movements of the main

chain containing catalytic aspartates Asp25/1025 of the

active site has been reported for the first time here

Thus the main chain containing these catalytic aspartates,

although having small B-factors, is very much adaptable to

the external stress induced by the cyclic urea scaffold This

observation suggests that it is important to distinguish such

adaptability from the concept of general flexibility arising

out of multiple conformations of the side chains and

thermal motions of the atoms therein

In the six cyclic urea complex only the substituents at the P2/P2¢ site are different as shown in Fig 2B While changes induced by cyclic urea inhibitors in the S1/S1¢ pocket are very similar (Fig 7), changes induced in the S2/S2¢ pocket depend on the substituents at the P2/P2¢ site These substituents point directly towards the polypeptide segment 27–32 of the S2/S2¢ subsite The allyl substituent at this site

in the case of XK216 is small and therefore there is not much change in the conformation of these residues (Fig 8) However, when a larger hydrophobic moiety like the naphthyl group, as in case of XK263, is present at the P2/P2¢

Fig 6 Superposition of cyclic urea bound HIV-1 protease in PDB file 1HWR (shown by green carbons) on unliganded HIV-1 protease (shown by yellow carbons) The relieved steric contacts are marked.

Fig 7 Superposition of cyclic urea inhibitor-bound HIV-1 protease (shown by green carbons) on the unliganded structure (shown by yellow carbons).

site, the main chain as well as the side chain of the

hydrophillic residue Asp30 moves away as shown in Fig 9

Further, when a hydrophilic group such as NH2replaces

one of the benzene rings of the naphthyl moiety above, as in

the case of DMP450, the aspartic acid residue Asp30 moves

toward the inhibitor and makes a hydrogen bond with this

new substituent (Fig 10)

More bulky groups at this site, as in SD146, causes steric

hindrance near Asp30 inducing the side chain of Asp30 to

move away (Fig 11)

Discussion and conclusion

Structure alignments are very often used to derive functional

information on proteins Distant evolutionary relationships

are also detected exclusively by structure alignments

Structural superpositions using complete proteins or

domains thereof, are a powerful method of identifying

ligand- or mutation-induced conformational changes The

inferences drawn from such comparisons are reliable

especially when the structures superimposed are variants

of the same protein crystallizing in isomorphous space groups, as is the case here Comparison of unliganded and ligand-bound HIV-1 protease is important for obtaining information about adaptability of residues in the active site The flaps in unliganded HIV-1 protease assume two very different conformations, open and closed, whereas the flaps always assume a closed conformation in ligand-bound structures When ligand-bound and open-flap unliganded HIV-1 protease structures are superimposed the Rmsd is greater than 1.0 A˚, with changes being distributed through-out the protein This might suggest that concerted changes distributed throughout the protein are needed to bring the flaps into a closed conformation Therefore, comparison of closed-flap unliganded structure with ligand-bound struc-tures would accurately reflect changes induced by ligand binding alone The uliganded structure we have determined

is of closed-flap conformation Comparison of the unligan-ded C95A mutant structure of HIV-1 protease with that complexed to cyclic urea-based inhibitors reveals that the residues which are known to be less flexible and are characterized by a small B-factor are not necessarily less

Fig 8 Superposition of XK216 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons).

Fig 9 Superposition of XK263 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons).

FEBS 2003 Adaptability and flexibility of HIV-1 protease (Eur J Biochem 270) 1237

adaptable to environmental changes Residues 23–26 in

both monomers of HIV-1 protease are considered to be very

‘robust’ and ‘rigid’ [2] The electron density of these

segments is also very well defined and they have a small

B-factor, implying less flexibility of this segment However

the present study shows that these residues are very

adaptable to internal or external stresses This property

may have been built into HIV-1 protease to cater to the

functional requirement that the enzyme cleave substrates of

eight different sequences [4] Cys95 at the dimer interface is

far away from the active site Also the B-factors of residues

91–99 are significantly more than those of segment 23–26

(Fig 3) Even so the C95A mutation does not cause much

change in the structure near the mutation site [3] Instead it

affects the catalytic site residues 23–26 thereby suggesting

that the residues 23–26 are more adaptable than residues

around 95 Hence it is important to distinguish this type of

adaptability from the concept of flexibility Zoete et al [2]

have analysed the X-ray structures of 73 HIV-1 protease

complexes to look at the overall structural variability of

protease Interestingly, they found that the pattern of structural variability of different residues in all of these structures is very much the same as the pattern of B-factors

of these residues in any single structure Thus the analysis of

an ensemble of structures gives information about the inherent flexibility of a protein However, the information about adaptability is lost Only analysing and comparing structures on a one to one basis would provide information about adaptability

Here in this paper we are trying to distinguish between the concept of flexibility and that of adaptability in protein structures Very often, these two terms are used inter-changeably in the literature However with the growing amount of structural information available about proteins, there arises a need to analyse and understand the structural features in a more comprehensive and accurate way One must have a well-defined tool to understand different properties of protein structure We thus feel that the word

‘flexibility’ with reference to protein structure should be reserved to describe the conformational variability of a Fig 11 Superposition of SD146 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons) Fig 10 Superposition of DMP450 bound HIV-1 protease (shown by green carbons) on the unliganded protease structure (shown by yellow carbons).

residue as well as to include the effects of thermal motions of

atoms therein On the other hand, the word ‘adaptability’

should be used to describe the ability of a residue/region to

adjust and accommodate itself in response to a stress/

change in its environment This stress could be either

internal, as in case of mutation of a nearby residue, or

external, as in case of presence of an inhibitor or other

nonprotein molecule in its surrounding This distinction is

important: we see in the present analysis that a less flexible

region need not necessarily be less adaptable

Acknowledgements

We thank the National Facility for Macromolecular Crystallography,

BARC for providing all the X-ray and biochemistry equipment used in

this investigation We are thankful to S K Sikka for encouragement

and support We thank K K Kannan, B Pillai and V Prashar for

scientific discussions and S.R Jadhav for technical help.

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