| CONJOINED RIGID BODY/TORSION ANGLE DYNAMICS | THE STRUCTURES WERE CALCULATED BY CONJOINED RIGID BODY/TORSION ANGLE DYNAMICS (SCHWIETERS & CLORE (2001) J.MAGN.RESON 152, 288-302; (CLORE & BEWLEY (2002) J.MAGN.RESON. 154, 329-335) THE TARGET FUNCTIONS COMPRISES TERMS FOR THE NOE RESTRAINTS, THE SIDECHAIN TORSION ANGLE RESTRAINTS, THE BACKBONE TORSION ANGLE RESTRAINTS FOR 4 VARIABLE REGIONS OF IIAMTL, THE DIPOLAR COUPLING RESTRAINTS (CLORE ET AL. J.MAGN.RESON. 131, 159-162 (1998); J.MAGN.RESON. 133, 216-221(1998)), THE RADIUS OF GYRATION (KUSZEWSKI ET AL. (1999), AND A QUARTIC VAN DER WAALS REPULSION TERM (NILGES ET AL. (1988) FEBS LETT. 229, 129- 136). THE STARTING COORDINATES COME FROM THE X-RAY STRUCTURES (WITH PROTONS ADDED) OF E. COLI HPR (1POH, JIA ET AL. (1993) J.BIOL.CHEM. 268, 22940-22501, RESOLUTION 1.5 A); AND IIAMTL (MOLECULE D OF 1A3A, VAN MONTFORT ET AL. STRUCTURE 5, 217-225 (1998); RESOLUTION 1.8A). SEVERAL DIFFERENT INITIAL ORIENTATIONS OF THE TWO PROTEINS WERE EMPLOYED WITH THE CA-CA DISTANCE BETWEEN THE ACTIVE SITE HISTIDINES RANGING FROM 28 TO 95 A, INCLUDING ORIENTATIONS WHERE THE TWO ACTIVE SITE HISTIDINES ARE NOT OPPOSED AND WHERE HPR IS DIRECTED TOWARDS THE FACE OF IIAMTL OPPOSITE TO THE IIAMTL ACTIVE SITE. THE BACKBONE COORDINATES AND NON-INTERFACIAL SIDECHAINS (EXCLUDING THE FOUR VARIABLE REGIONS OF IIAMTL: RESIDUES 51-54, 66-78, 91-96 AND 104-110) ARE TREATED AS RIGID BODIES THROUGHOUT WITH IIAMTL HELD FIXED, HPR ALLOWED TO ROTATE AND TRANSLATE, AND THE AXIS OF THE DIPOLAR COUPLING ALIGNMENT TENSOR FREE TO ROTATE. THE INTERFACIAL SIDECHAINS, AS WELL AS THE BACKBONE AND SIDECHAINS OF THE FOUR VARIABLE REGIONS OF IIAMTL, ARE GIVEN FULL TORSIONAL DEGREES OF FREEDOM. ALSO NOTE THAT GLU59 AND HIS111 ARE REFINED IN TWO ALTERNATE CONFORMATIONS.
IN THIS ENTRY THE LAST COLUMN REPRESENTS THE AVERAGE RMS
DIFFERENCE BETWEEN THE INDIVIDUAL SIMULATED ANNEALING
STRUCTURES AND THE MEAN COORDINATE POSITIONS. IT IS
IMPORTANT TO NOTE THAT THE VALUES GIVEN FOR THE BACKBONE
ATOMS AND NON-INTERFACIAL SIDECHAINS (EXCLUDING
THE FOUR VARIABLE REGIONS OF IIAMTL) PROVIDE ONLY A
MEASURE OF THE PRECISION WITH WHICH THE RELATIVE
ORIENTATION OF THE TWO PROTEINS HAVE BEEN DETERMINED AND
DOES NOT TAKE INTO ACCOUNT THE ERRORS IN THE X-RAY
COORDINATES OF HPR AND IIAMTL.
RESIDUE NUMBERING:
IIAMTL: 4-147 (RESIDUES 1-3 ARE DISORDERED
IN SOLUTION AND NOT VISIBLE IN THE ELECTRON DENSITY
MAP OF THE CRYSTAL STRUCTURE OF THE FREE PROTEIN).
HPR: 301-385 (CORRESPONDING TO RESIDUES 1-85).
PHOSPHATE: RESIDUE 200
THREE SETS OF COORDINATES ARE GIVEN:
MODEL 1: RESTRAINED REGULARIZED MEAN
COORDINATES OF THE UNPHOSPHORYLATED HPR-IIAGLC COMPLEX
SOLVED ON THE BASIS OF 107 INTERMOLECULAR
INTERPROTON DISTANCE DISTANCE RESTRAINTS, 105 INTRAMOLECULAR
DISTANCE RESTRAINTS (RELATING TO INTERFACIAL SIDECHAINS, AS
WELL AS THE FOUR VARIABLE REGIONS OF IIAMTL), 70 INTERFACIAL
SIDECHAIN TORSION ANGLE RESTRAINTS, 62 TORSION ANGLE
RESTRAINTS FOR THE VARIABLE REGIONS OF IIAMTL, AND 528
RESIDUAL DIPOLAR COUPLINGS.
CROSS-VALIDATION
WAS USED FOR THE DIPOLAR COUPLINGS (CLORE AND GARRETT
(1999) J. AM. CHEM. SOC. 121, 9008-9012).
MODEL 2: RESTRAINED REGULARIZED MEAN COORDINATES FOR THE
MODEL OF THE DISSOCIATIVE PHOSPHORYL TRANSITION STATE
HPR-IIAMTL COMPLEX. EXPERIMENTAL RESTRAINTS ARE
IDENTICAL TO THOSE USED FOR MODEL 3, BUT COVALENT
GEOMETRY RESTRAINTS ARE INCLUDED RELATING TO THE
PENTACOORDINATE PHOSPHORYL GROUP IN A TRIGONAL BIPYRAMIDAL
GEOMETRY. THE STRUCTURE IS DERIVED FROM
MODEL 3 BY RESTRAINED MINIMIZATION. THE N-P BOND LENGTHS
ARE RESTRAINED TO 3 A. THE CA-CA DISTANCE BETWEEN HIS315
(HPR) AND HIS65 (IIAMTL) REMAINS ESSENTIALLY UNCHANGED
FROM MODEL 3, BUT THE ND1-NE2 DISTANCE BETWEEN HIS315 AND
HIS65 IS REDUCED TO 6 A, WITH ESSENTIALLY IDEALIZED
GEOMETRY OF THE PHOSPHORYL TRANSITION STATE.
THE ND1-NE2 DISTANCE CORRESPONDS TO A DISSOCIATIVE
TRANSITION STATE. THE RMS DIFFERENCE BETWEEN THE MEAN
STRUCTURE OF THE UNPHOSPHORYLATED COMPLEX (MODEL 3)
AND THE TRANSITION STATE COMPLEX IS 0.2 A FOR
BACKBONE COORDINATES IMMEDIATELY ADJACENT TO THE ACTIVE
SITE HISTIDINES (RESIDUES 64-66 AND RESIDUES 316-317).
THE REMAINING BACKBONE COORDINATES DO NOT SHIFT.
MODEL 3: RESTRAINED REGULARIZED MEAN COORDINATES FOR THE
MODEL OF THE ASSOCIATIVE PHOSPHORYL TRANSITION STATE
HPR-IIAGLC COMPLEX. CALCULATED LIKE MODEL 2 BUT
WITH THE N-P BOND LENGTHS RESTRAINED TO 2A.
THE STRUCTURE IS DERIVED FROM
MODEL 1 BY RESTRAINED MINIMIZATION.
THE RMS DIFFERENCE BETWEEN THE MEAN
STRUCTURES OF THE UNPHOSPHORYLATED COMPLEX (MODEL 1)
AND THE TRANSITION STATE COMPLEX IS 0.4 A FOR
BACKBONE COORDINATES IMMEDIATELY ADJACENT TO THE ACTIVE
SITE HISTIDINES (RESIDUES 64-66 AND RESIDUES 316-317).
THE REMAINING BACKBONE COORDINATES DO NOT SHIFT.
HPR-IIAMTL COMPLEX
DEVIATIONS FROM IDEALIZED GEOMETRY:
BONDS 0.006 A, ANGLES 0.82 DEG, IMPROPER TORSIONS 0.97 DEG
RMS DEVIATIONS FROM NOE DISTANCE RESTRAINTS: 0.007 A
RMS DEVIATIONS FROM SIDECHAIN TORSION ANGLE RESTRAINTS:
0.26 DEG.
RMS DEVIATIONS FROM BACKBONE TORSION ANGLE RESTRAINTS:
1.2 DEG.
DIPOLAR COUPLING R-FACTORS (CLORE AND GARRETT (1999)
J. AM. CHEM. SOC. 121, 9008-9012):
HPR IIAMTL
NH 19.1% 19.2%
CaH 25.9% 18.7%
NC' 34.0% 32.1%
[NOTE ONE ALIGNMENT TENSOR IS USED FOR THE
NH DIPOLAR COUPLINGS (FOR BOTH HPR AND IIAMTL),
AND ANOTHER FOR THE
CAH AND NC' DIPOLAR COUPLINGS (FOR BOTH
HPR AND IIAMTL), SINCE THE LATTER SET OF DIPOLAR
COUPLINGS WERE OBTAINED
FROM A DIFFERENT BATCH OF PEG/HEXANOL
THAN THE FORMER. THE ORIENTATION OF THE
TWO ALIGNMENT TENSORS DIFFERS BY ONLY 1.9 DEG.
NOTE THE ALIGNMENT TENSORS FOR HPR
AND IIAMTL ARE THE SAME. FOR REFERENCE
THE DIPOLAR COUPLING R-FACTORS FOR THE FREE STRUCTURES
(USING INDIVIDUAL ALIGNMENT TENSORS FOR THE TWO PROTEINS)
ARE 21.3% (NH), 21.1% (CaH), 33.6% (NC') FOR
THE X-RAY STRUCTURE OF HPR, AND
19.2% (NH), 18.0% (CaH) AND 32.0% (NC') FOR THE RESTRAINED
REGULARIZED MEAN STRUCTURE OF IIAMTL IN THE COMPLEX]. | X-PLOR_NIH (HTTP://NMR.CIT.NIH.GOV/XPLOR_NIH) |
