| 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). THE TARGET FUNCTION COMPRISES TERMS FOR THE DIPOLAR COUPLING RESTRAINTS (CLORE ET AL. J.MAGN.RESON. 131, 159-162 (1998); J.MAGN.RESON. 133, 216- 221(1998)), INTERMOLECULAR NOE RESTRAINTS AND TORSION ANGLE RESTRAINTS. THE NON-BONDED TERMS INCLUDE A QUARTIC VAN DER WAALS REPULSION TERM (NILGES ET AL. (1988) FEBS LETT. 229, 129-136), RADIUS OF GYRATION RESTRAINTS
(KUSZEWSKI ET AL. (1999) J.AM.CHEM.SOC 121, 2337-2338) AT THE PROTEIN-PROTEIN AND PROTEIN-DNA INTERFACES, AND DATABASE TORSION ANGLE AND BASE-BASE POSITIONAL POTENTIALS OF MEAN FORCE (KUSZEWSKI ET AL. (2001) J.AM.CHEM.SOC 123, 3903-3918; CLORE & KUSZEWSKI (2003) J.AM.CHEM.SOC. 125, 1518-1525). THE STARTING COORDINATES FOR THE POUHD AND POUS DOMAINS OF OCT1 ARE TAKEN FROM THE 1.9 A RESOLUTION CRYSTAL STRUCTURE OF THE OCT1/MORE-DNA COMPLEX (1E3O) AND PLACED IN THE ORIENTATION OF THE 2.7 A RESOLUTION CRYSTAL STRUCTURE OF THE OCT1/PORE-DNA COMPLEX (1HFO) (REMENYI ET AL. (2001) MOL.CELL 8, 569-580). THE STARTING COORDINATES FOR SOX2 ARE DERIVED FROM THE NMR STRUCTURE OF THE RELATED BINARY SRY-DNA COMPLEX (1J46) (MURPHY ET AL. (2001) J.MOL.BIOL. 312, 481-499). THE STARTING COORDINATES FOR THE 19MER DNA WERE BUILT AS FOLLOWS: THE POUS AND POUHD HEMI-BINDING SITES (B.P. 11-14 AND 17-19, RESPECTIVELY) WERE DERIVED FROM THE 1.9 A RESIOLUTION STRUCTURE OF THE OCT1/MORE-DNA COMPLEX (1E3O); THE
SOX2 BINDING SITE (B.P. 1-10) WAS DERIVED FROM THE NMR STRUCTURE OF THE BINARY SRY/DNA COMPLEX (1J46); AND THE INTERVENING SEQUENCES (B.P. 15-16) AND REGIONS CONTAINING SUBSTITUTIONS (B.P. 1, 4 AND 10) WERE DERIVED FROM CLASSICAL DNA. THE RESULTING MODEL WAS SUBJECTED TO REGULARIZATION. THE STRATEGY USED IN THE CONJOINED RIGID BODY/TORSION ANGLE DYNAMICS CALCULATIONS IS AS FOLLOWS: THERE ARE 4 RIGID BODIES: (1) BACKBONE AND NON-INTERFACIAL SIDE CHAINS OF POUHD + B.P. 17-19 OF THE DNA (2) BACKBONE AND NON-INTERFACIAL SIDE CHAINS OF POUS; (3) BACKBONE AND NON-INTERFACIAL SIDE CHAINS OF SOX2 + B.P. 1-4 OF THE DNA; (4) THE AXIS OF THE DIPOLAR COUPLING ALIGNMENT TENSOR. RIGID BODIES 1-3 HAVE ROTATIONAL AND TRANSLATIONAL DEGREES OF FREEDOM, WHILE RIGID BODY 4 IS GIVEN ONLY ROTATIONAL DEGREES OF FREEDOM. THE FOLLOWING SIDE CHAINS WERE GIVEN TORSIONAL DEGREES OF FREEDOM: (1) POUHD: 10 RESIDUES AT POUHD-DNA INTERFACE (RESIDUES 107, 108, 113, 144, 147, 148, 151, 154, 155 AND 158) WITH 24 SIDE C
AIN TORSION ANGLES RESTRAINED TO WITHIN A RANGE OF +/-20 DEGREES OF VALUES IN BINARY OCT1/DNA COMPLEXES (2) POUS: (A) 6 RESIDUES AT POUS/SOX2 INTERFACE (RESIDUES 14, 17, 18, 21, 26 AND 52); (B) 14 RESIDUES AT POUS/DNA INTERFACE (RESIDUES 20, 27, 41, 42, 44, 45, 46, 48, 49, 54, 58, 59, 62 AND 63) WITH 35 SIDE CHAIN TORSION ANGLES RESTRAINED TO WITHIN A RANGE OF +/-20 DEGREES OF VALUES IN BINARY OCT1/DNA COMPLEXES (3) SOX2: (A) 7 RESIDUES AT POUS/SOX2 INTERFACE (RESIDUES 59, 62, 63, 66, 67, 71,
73); (B) 18 RESIDUES AT SOX2/DNA INTERFACE (RESIDUES 4, 6, 7, 8, 9, 10, 12, 13 17, 31, 35, 43, 44, 51, 55, 76, 78, 79) WITH 35 SIDE CHAIN TORSION ANGLES RESTRAINED TO WITHIN A RANGE OF +/-20 DEGREES OF VALUES IN BINARY SRY/DNA COMPLEXES. BASE PAIRS 5-16 OF THE DNA WERE GIVEN TORSIONAL DEGREES OF FREEDOM WITH 220 LOOSE BACKBONE PHOSPHODIESTER TORSION ANGLE RESTRAINTS TO PREVENT LOCAL MIRROR IMAGES (MURPHY ET AL. (2001) J.MOL.BIOL. 312, 481-499).
THE NUMBERING SYSTEM IS AS FOLLOWS:
OCT1 POUS DOMAIN: 5-79
OCT1 POUHD DOMAIN: 110-163
SOX-2 HMG-BOX: 206-282
RESIDUES 1-4, 80-109, 201-205 AND 283-288 ARE DISORDERED
IN SOLUTION AND THUS NOT INCLUDED IN THE COORDINATES.
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 SINCE THE BACKBONE AND NON-
INTERFACIAL SIECHAINS OF THE THREE PROTEIN DOMAINS ARE
TREATED AS RIGID BODIES, THESE NUMBERS DO NOT TAKE INTO
ACCOUNT THE ERRORS IN THE X-RAY COORDINATES OF OCT1 OR
THE NMR COORDINATES OF THE HOMOLOGOUS SRY.
RESIDUE NUMBERING: THIS FOLLOWS THE NUMBERING USED
PREVIOUS STRUCTURAL WORK ON THE BINARY OCT1/DNA
COMPLEX (KLEMM ET AL. (1994) CELL 77, 21-32;
REMENYI ET AL. MOL.CELL (2001) 8, 569-580);
AND THE BINARY SRY/DNA COMPLEX (MURPHY ET AL.
(2001) J.MOL.BIOL. 312, 481-499).
THE SIDECHAINS OF K18, Q22, K262, R265 AND K276 ARE IN
MULTIPLE CONFORMATIONS.
EXPERIMENTAL NMR RESTRAINTS:
RESIDUAL DIPOLAR COUPLINGS: 345
(1) SOX2: 51 NH, 39 NC', 49 CaC'
(2) POUS: 39 NH, 33 NC', 34 CaC'
(3) POUHD: 39 NH, 34 NC', 27 CaC'
INTERMOLECULAR NOE-DERIVED INTERPROTON DISTANCE
RESTRAINTS:
67 (16, 48 AND 3 at POUS/SOX2, SOX2/DNA
AND POUHD/DNA INTERFACES)
TORSION ANGLE RESTRAINTS: 21
(18 AT POUS/SOX2 INTERFACE AND 3 AT SOX2/DNA
INTERFACE).
NH DIPOLAR COUPLING R-FACTORS
TERNARY COMPLEX INDIVIDUAL DOMAINS
SOX2 17.7% 16.5%
POUS 16.7% 16.2%
POUHD 17.7% 17.5%
(THE VALUES GIVEN FOR THE INDIVIDUAL DOMAINS
ARE CALCULATED USING A SEPARATED ALIGNMENT
TENSOR FOR EACH DOMAIN AND ARE SIMPLY LISTED
FOR REFERENCE. THE VALUES FOR THE TERNARY
COMPLEX (USING THE RESTRAINED REGULARIZED MEAN
COORDINATES) MAKE USE OF A SINGLE ALIGNMENT
TENSOR FOR THE ENTIRE COMPLEX).
NON-EXPERIMENTAL RESTRAINTS:
(1) 220 LOOSE TORSION ANGLE RESTRAINTS FOR THE
SUGAR-PHOSPHATE BACKBONE
(2) 106 LOOSE TORSION ANGLE RESTRAINTS FOR SIDE CHAINS
AT PROTEIN-DNA INTERFACES
(3) 35 LOOSE DISTANCE RESTRAINTS AT THE POUS/DNA AND
POUHD/DNA INTERFACES TO PRESERVE HYDROGEN BONDING
INTERCATIONS AND SALT BRIDGES TO BASES AND PHOSPHATES
(4) WEAK NCS RESTRAINT TO PROVIDE A TRANSLATIONAL RESTRANT
BETWEEN POUS AND POUHD. | X-PLOR NIH |
