[Identification of variable regions in HIV1-PR molecules]



In identification of variable regions in HIV1-PR moleclules, the D2 code (ProteinEncoder) is compared with two existing methods: a (Phi, Psidihedral angle-based method and the PB block method (structural alphabet).


(1) About HIV1-PR

For comparative performance analysis, we use 72 crystal structures (142 chains) of the N37S mutant and the 28 NMR models (56 chains) of a mutant (1bve).
Variable regions are identified as the region where more than one type of D2 codes or PB blocks are assigned.

Shown below are their amino acid sequences:
 
         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
N37S mutant

1bve

PQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF
  x                                                                                           x
PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGATLNF
                        <=> Active site (Res.25-27)

[MEMO] The list of the N37S mutants: click_here. Among the 72 PDB entries, the chains of 1hxb are a {I3V, N37S} mutant. Their amino acid sequence is the following:
                                PQVTLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMSLPGRWKPKMIGGIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNF
[MEMO] 1bve is a {I3V, N37S, C95A} mutant.



Shown below are superimposition of the structures, which are the 'master-slave' alignments by DALI with 1d4jA (or model26A for NMR models) as master:

jpg image
jpg image
jpg image
jpg image
jpg image
jpg image
jpg image
jpg image
(A) N37S (P21212) (B) N37S (P212121) (C) N37S (P61) (D) 1bve (NMR)
'Master-slave' alignments by DALI, where the master is 1d4jA for crystal structures and model26-A for NMR models.
In the figure, top and front views are given, where yellow spheres indicate the position of the residues that assume a "minority" D2 code (i.e., D2-variable residues).
(A) 49 crystal structures (98 chains) of space group P21212 of the N37S mutant.
(B) 12 crystal structures (24 chains) of space group P212121 of the N37S mutant.
(C) 9 crystal structures (18 chains) of space group P61 of the N37S mutant.
(D) 28 NMR models (56 chains) of a mutant.




(2) Averaged structure of HIV1-PR

(2.1) Most frequentry occured D2 code assignment

         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
N37S mutant

NMR models

--001R00R000000RRG00000R0QBGR000000000000000000000R000000000R00000RRG000000000R0R00000GAAAAABR000--
    x                                                                                 x
--00RR00
R000000RRG00000R0QBGR000000000000000000000R000000000R00000RRG000000000R0R00000QAAAAABR000--
                        <=> Active site
*You could use program NtileCodePredictor to obtain the most frequentry occured D2 code assignment:

          % NtileCodePredictor -t frag_code5_HIV1PR_ID.tbl    filename.pdb
 
  Conversion table frag_code5_HIV1PR_ID.tbl  (sample), which is compiles from the 142 chains of the N37S mutan, is available from the PROGRAM>NtileCodePredictor>DOWNLOADS section.
*See the bottom for description of the D2 codes.

[MEMO] NtileCodePredictor computes the most frequentry occurred D2 code (the major D2 code) for each five-residue fragment based on the "fragment-to-D2 code" conversion table.



(2.2) Most frequentry occured PB block assignment

         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
N37S mutant

NMR models
ZZDFKBCCDDDDDEEHIACDDDDDFKBMBDCDDDDDDEHJACDDDDDFKNOIACDDDDEHIACDEEHIACDFBLCDFKLCBDCDFKLMMMMNOPACDZZ
  xxxxxxx     x            xxxxx  xxxx  xx x    xx    xxxx
ZZFKLMBLCDDDDEHHIACDDDDDFKBCKLPCDDFBLCHJMMDFDDDFBGOIACFBLCEHIACDEEHIACDFBLCDFKLCBDCDFKLMMMMNOPACDZZ
                        <=> Active site
*See the bottom for description of PB blocks.


(2.3) Average of (Phi, Psi)-dihedral angles

1 2 3 4  ...  39 40 41  ...  72 73 74  ...  97 98 99
N37S(PHI)
N37S(PSI)

NMR(PHI)
NMR(PSI)
---
135

---
135
-112
123

-86
134
-112
122

-132
114
-83
165

-110
-28
...
...

...
...
-77
149

-80
35
98
-49

18
-54
-87
156

2
84
...
...

...
...
-132
126

-95
144
-33
-165

-79
-98
-78
135

-96
104
...
...

...
...
-100
136

-91
98
-136
137

-104
126
-162
---

-139
---
*Dihedral angles are calculated in degrees, using the DSSPcont server.[2]
*The values are average over the 142 chains in the case of the N37S mutant, and the 56 chains in the case of the NMR models of 1BVE.




(3) Identification of variable regions in HIV1-PR

The bar graphs below show the spatial distribution of the occurrence of (1) large deviation of dihedral angles from the average, (2) minority D2 code assignment (i.e., D2-variable residues), and (3) minority PB-block assignment (i.e., PB-variable residues).

The first row shows the residues whose phi or psi angle is away from the average by more than 30°.
Recall that, due  to  the anticorrelation between Phi and Psi anglesdihedral angle fluctuations may represent a change not in backbone conformation but in side-chain reorientation. [1]
Crystal Structures: The phi or psi angles of residues 40, 50, 51, 73, 78, and 86 are almost always deviated from the average more than 30° (See (4) Discussion below for detailed discussion).
NMR models: Variable regions of the NMR models are more widely distributed than that of crystal structures.

The second row shows spatial distributions of the occurrence of minority D2 code assignment.
Crystal Structures: 348 residues of the 142 chains are assigned a minority D2 code. 40% of the minority D2 codes are assinged at residue 50 or 51 and 15% are assigned at residue 5. The deformations are provably explained by crystal packing and variation of the size of the ligands bound.
NMR models: 480 residues of the 56 chains are assigned a minority D2 code. 22% of the minority D2 codes are assinged at the loop around residue 40 and 10% are assinged at the loop around residue 60. The deformations are provably explained by collision with another molecule. Note that no minority D2 code is assinged at the loop around residue 50.

The third row shows spatial distributions of the occurrence of minority PB-block assignment.
Crystal Structures: Not only around residue 50, but also residue 30, 40, and 71-80 often assume minority PB block.
NMR models: Variable regions of the NMR models are more widely distributed than that of crystal structures.



Crystals of the N37S mutant NMR models (1bve)
Residue with
dPHIi > 30°  or
dPSIi > 30°
jpg image
jpg image
D2-variable
residue
jpg image
jpg image
PB-variable
residue
jpg image
jpg image

 
* [Definition] dPHIi := PHIi - <PHIi>, where PHIi is the phi dihedral angle of the i-the residue and <PHIi> is the average over the chains (142 chains in the case of the N37S mutant, and 56 chains in the case of the NMR models of 1BVE).  dPSIi is also defined similarly. That is, the first row shows the residues whose phi or psi angle is away from the average by more than 30°.

[MEMO] Gray bars and dark gray bars in the background indicate the position of minor and major drug resistance mutations, respectively.




(4) Discussion

The phi or psi angles of residues 40, 50, 51, 73, 78, and 86 are almost always deviated from the average more than 30°. In particular, the phi or psi angles of residues 40 and 73 are almost always deviated from the average more than 110° because chain A and chain B of the HIV-1 PR dimer have different average values (for example, X and X + 340) on these residues (Data not shown).

Recall that, because of periodicity, the dihedral angles X and X+360° are identical. Therefore, the difference between two dihedral angles X and X+340° is 20°, which means the HIV-1 PR molecules have similar local structures around the residues.

As an example, let's consider the local structure of 1ajx chain A and chain B around residue 73 (Figure 4.1 bottom). The phi angles of residue 73 of 1ajxA and 1ajxB are 177.6° and -170.2° respectively. Because of periodicity, the difference between them is 12.2°. Figure 4.1 bottom is the result of the alignment computed by the DALI server, which shows fragments from residue 58 to residue 88 of 1ajxA and 1ajxB are in very similar conformations (RMSD 0.3Angstrom). The fragments have an identical D2 code sequence but different PB block assignments: "Ccap-beta-beta-Ncap" and "beta-Ccap-Ncap-alpha" around residue 73. 

On the other hand, because of anticorrelation, a big change in dihedral angles does not necessary mean a change in the Ca backbone, but a reorientation of the peptide in question. [1]

As an example,
let's consider the local structure of 1ajx chain A and chain B around residue 40 (Figure 4.1 top). The phi angles of residue 40 of 1ajxA and 1ajxB are 133.7° and 101.5° respectively, that is, their difference is 32.2°. The difference of psi angles is 15.0°.  Figure 4.1 top is the result of the alignment computed by the DALI server, which shows fragments from residue 25 to residue 55 of 1ajxA and 1ajxB are also in very similar conformations (RMSD 0.5Angstrom). The fragments have an identical D2 code sequence around residue 40 but different PB block assignments: "coil-Ncap-beta" and "coil'-Ncap-Ncap'" around residue 41. 




jpg image
Residue 40


jpg image
Residue 73
< Around res. 40>
39 PRO A      -84.3  137.6  175.5
40 GLY A      133.7  175.5  178.0
41 ARG A      -86.4  147.5  176.9

39 PRO B      -88.8  150.0  176.9
40 GLY B      101.5 -169.5  180.0
41 ARG B      -90.1  156.3  176.8

< Around res. 73>
72 ILE A     -123.8  139.4  177.8
73 GLY A      177.6 -177.8  178.1
74 THR A      -77.1  144.8 -179.6

 72 ILE B     -132.3  115.5  179.8 
73 GLY B     -170.2 -161.5 -177.2
74 THR B      -80.0  129.3 -179.3
jpg image
Around res. 40
jpg image
Around res. 73
jpg image
Around res. 40
jpg image
Around res. 73
(A) Location of residue 40 and 73 (B) Dihedral angles  (phi,psi,omega) of  1ajxA and 1ajxB (C)  1ajxA and 1ajxB (D) NMR models
Figure 4-1.
(A)
 Location of residue 40 and 73.
(B) Dihedral angles  (phi,psi,omega) of 1ajxA and 1ajxB.
(C) Alignments of backbone fragments of 1ajxA and 1ajxB: residues 35-45 (top) and residues 68-78 (bottom). They are the 'master-slave' alignments by DALI with 1d4jA as master. 1d4jA is not shown in the figure.
(D) The 'master-slave' alignments of fragments of 56 NMR chains by DALI with model26-A as master: residues 35-45 (top) and residues 68-78 (bottom). Residues are colored according to thier D2 code:  '0' in blue, 'A' in red, 'B' and 'Q' in orange, 'G' in green, and 'R' in yellow. 




DALI pairwise alignment:
DALI score DALI alignment
 Fragment res.25-55
of
1ajxA and 1ajxB
Z-value: 4.9
RMSD: 0.5
LALI: 31
NRES: 31
%ID: 100

jpg image
 Fragment res.58-88
of
1ajxA and 1ajxB
Z-value: 5.8
RMSD: 0.3
LALI: 31
NRES: 31
%ID: 100

jpg image
Figure 4-2.
(1) DALI score shows values returned by the DALI server, where
    Z-value: Z-score of the match
    RMSD: RMSD of the match
    LALI: Number of aligned positions
    NRES: Number of residues in matched structure
    %ID: Sequence identity of aligned positions.
(2) DALI alignment shows alignment of fragment pairs computed by the DALI server: res.25-55 of 1ajxA and 1ajxB (top) and res.58-88 of 1ajxA and 1ajxB (bottom).
    Large black spheres in the figure are residue 40 and 73 of 1ajxA.
   
Large white spheres in the figure are residue 40 and 73 of 1ajxB.

[NOTE] Chains shorter than 30 residues are ignored by the DALI server.


D2 code assignment:
         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
1ajxA

1ajxB

--00RR00R000000RRG00000R0QBGR000000000000000000000R000000000R00000RRG000000000R0R00000QA2AAABR000--
    x                                            x                                      x
--00
1R00R000000RRG00000R0QBGR00000000000000000000QR000000000R00000RRG000000000R0R00000QAAAAABR000--
                        <=> Active site


PB block assignment:
         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
1ajxA

1ajxB

ZZDFKBCCDDDDDEEHIACDDDDDFKBMBACDDDDDDEHIADDDDDDFBGOIACDDDDEHIACDEEHIACEHJACDFKLCBDCDFKLMMMMNOPACDZZ
                                       x x      xx x                  xxxx   x
ZZDFKBCCDDDDDEEHIACDDDDDFKBMBACDDDDDDEHJACDDDDDFKNOPACDDDDEHIACDEEHIACDFBLCDFBLCBDCDFKLMMMMNOPACDZZ

                        <=> Active site


(Phi, Psi, Omega)-dihedral angles:
1 2 3 4  ...  35 36 37 38 39 40 41 42 43 44 45  ...  68 69 70 71 72 73 74 75 76 77 78  ...  97 98 99
PHI(1ajxA)
PSI(1ajxA)
Omega(1ajxA)

PHI(1ajxB)
PSI(1ajxB)
Omega(1ajxB)
---
153
178

---
170
178
-115
111
-176

-115
135
-178
-103
118
173

-122
129
-179
-74
159
-174

-95
173
-175
···
···
···

···
···
···
-42
125
178

-43
124
179
-163
167
-180

-161
172
179
-113
134
-180

-103
129
180
-122
157
180

-126
161
-180
-84
138
176

-89
151
177
134
175
178

101
-170
180
-86
148
177

-90
156
177
-135
161
-177

-130
159
-180
-124
140
179

-120
156
-180
-74
149
-180

-85
148
-180
-151
155
176

-154
146
-179
···
···
···

···
···
···
85
-8
178

-84
-4
179
-105
119
177

-100
126
179
-66
149
177

-86
124
178
-151
149
179

-124
155
176
-124
139
178

-133
116
-180
178
-178
178

-170
-162
-177
-77
145
-180

-80
129
-179
-136
130
177

-128
138
178
-103
135
180

-108
145
-179
-121
124
180

-124
131
-178
-173
-167
-179

-175
-175
-179
···
···
···

···
···
···
-104
133
-178

-104
136
-179
-136
129
177

-133
130
174
-153
---
---

-151
---
---
*http://swift.cmbi.ru.nl/servers/html/index.html




<FOR REFERENCE>

SA-Encode (SA-27):
         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
1ajxA

1ajxB

 NMPOIYHSXKNMXUFSLXLXKPRGBQPILNTKYXLTLJJNMLXMNNXFCRJTLXLLNNUSLXMTFFQLXMJJNMNJFSPIKNMNYEZZWVWDSXLX
    x           x    x       xx    xxx    x   x xx     xxxx     xx x     x x          x xxxx    x
 NMPEIYHSXKNMXUFQLXLXLPRGBQPIKTTKYXKXJJJNMKXMNLXYZRJTLXKNTLUSLXMNUFSLXMJJTMMJFSPIKNMNYBZWAAADSXLN

                        <=> Active site
*http://bioserv.rpbs.jussieu.fr/cgi-bin/SA-Encode

DSSP assignment:
         1         2         3         4         5         6         7         8         9        10
1234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890
1ajxA

1ajxB

    TTT  SSSSSSTTSSSSSSS TT TT SS T   TT  SSSSSSSTTSSSSSSSSSSSSSSSTTSSSSSSSSST  TT SS HHHHTTTT
 
    TTT  SSSSSSTTSSSSSSS TT TT SS T   TT  SSSSSSSTTSSSSSSSSSSSSSSSTTSSSSSSSSST  TT SS HHHHTTTT

                        <=> Active site
*http://swift.cmbi.ru.nl/servers/html/index.html




References
  1. McCammon  JA,  Gelin  BR,  Karplus  M. 1977. Dynamics of folded  proteins. Nature 267:585-590.
  2. Carter P, Andersen CA, Rost B. DSSPcont: Continuous secondary structure assignments for proteins. Nucleic Acids Res. 2003 Jul 1;31(13):3293-5.



[The D2 codes]


[The PB label]

[The DSSP code]