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IV. ASSIGNMENT OF THE RESONANCES OF THE INHIBITOR AND NOES BETWEEN THE PROTEIN AND THE INHIBITOR

IV. ASSIGNMENT OF THE RESONANCES OF THE INHIBITOR AND NOES BETWEEN THE PROTEIN AND THE INHIBITOR

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Figure 5 Part of a 2-D 1H, 15N HMQC spectrum of inhibited stromelysin-1

where the ring nitrogens of His resonate. The delay where antiphase magnetization evolves is set to 22 ms thus favoring the weak two- and three-bond

couplings of Ny1 and Nq2 to the protons of Cy2H and Cq1H [41]. The

deprotonated (h-type) nitrogen typically resonates near 249 ppm. These

resonances for inhibited sfSTR are near 200 to 210 ppm, shifted upfield by

ligation to the zinc ions. For the stable Ny1-H tautomer two strong couplings are

observed from the deprotonated Nq2 nucleus to the Cy2H and Cq1H protons. For

the stable Nq2-H tautomer only one strong coupling is observed from the

deprotonated Ny1 nucleus to the Cq1H proton. For the imidazolium tautomer the

resonances of the ring nitrogens are both near 176 ppm and equivalent couplings

from these nitrogens to both Cy2H and Cq1H protons are observed. For inhibited

sfSTR, His-151, -166, -201, -205 and -211 are in the Ny1-H tautomer, His-179 is

in the Nq2-H tautomer and His-96 and -224 are in the imidazolium tautomer.

Specific labeling of the Ny1 nucleus supports these assignments [5]. (From Ref. 5.)



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 6 X-half filters used for filtering or selecting 13C and 15N-attached

protons. Thick and thin closed rectangles are 180j and 90j pulses, respectively,

open rectangles are spin lock pulses. (A) A simple X-half filter (2). The delay H is

equal to (1/(2[1JXH]) where 1JXH is the one-bond coupling between proton and

either 13C (120 to 140 Hz) or 15N (95 Hz). The second 90j pulse is the editing



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



V. STRUCTURE CALCULATIONS

Peak intensity data from NOE experiments were accumulated and converted to interproton distances by calibrating against the expected short

distances in secondary structure elements. These data were complimented

with coupling constants determined from the 3-D HNHA and HNHB

experiments. A total of 2589 peaks were assigned in all NOE experiments.

After removal of nonconstraining and ambiguous NOEs, typically found

in mobile regions, 1814 meaningful restraints remained: 325 intraresidue,

429 sequential, 324 short-range (i+2 to i+5), 665 long range ( > i+5), and

71 intermolecular. Using a gridsearch program [44] 379 dihedrals (140 f,

140 c, 99 m1) were generated from sequential and intraresidue NOEs and

coupling constant data from HNHA and HNHB experiments. Structures

were calculated using the variable target function algorithm DIANA [45],

but it should be noted that in recent years this method has been replaced by

torsion angle dynamics methods that are far more efficient [46,47]. To

determine the structure of the complex, a residue template of the inhibitor

was built as a single residue covalently linked through an oxygen of the

carboxylate moeity of the inhibitor (Fig. 1) to the zinc which was

covalently bonded to the Nq2 of His-201. The residue template of His151 was created with the structural zinc covalently attached to its Nq2

atom. The structure calculation process is largely iterative with trial structures calculated and incompatible NOEs reassigned or removed and new

NOEs assigned on the basis of agreement with the trial structure. In the

final calculations, and to reduce bias in structure selection, plots of rmsd

and number of structures versus target function [48] of the final 80



pulse. The phase cycling of this pulse with respect to the receiver determines

whether X-nucleus attached protons are selected or filtered. If both signals are

added to the receiver (x,x) X-nucleus attached protons are filtered; and if the

receiver phase is alternated (x,-x) the X-nucleus attached protons are selected. (B)

A doubly tuned half filter for filtering 13C attached protons [42]. In this experiment the filter consists of two delays (H 1,H 2) tuned to different 1JCH values

resulting in superior suppression of artifacts. (C) A doubly tuned time-shared half

filter for 13C/15N (43). In this experiment D = 1/(41JNH), D1 = 1/(41JCH) and D2 =

[1/(41JNH À 1/(41JCH)]. Phase cycling the receiver selects or filters both 13C and

15

N attached protons.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 7 2-D 13C doubly filtered NOESY of inhibited sfSTR using the X-half

filters of Fig. 4A and B. The NOE correlations of the rings of the P1V and the P3V

group are shown. The Hh and Hg protons of the P1V group were distinguished in a

similar 2D 13C doubly filtered TOCSY. The specific assignment of the protons of

the P3V group were determined by NOEs between the H2,6 and the NH of the P3V

in 2D 13C,15N-filtered experiments using the time-shared doubly tuned half filter

of Fig. 4C.



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Figure 8 Sections of a 3-D 13C-separated, 13C-filtered NOESY of inhibited

sfSTR. Only NOEs between the 12C-attached protons of the inhibitor and the

13

C-attached protons of the protein are observed in this spectrum. These NOEs

describe the S1V, S2V and S3V subsites of sfSTR. Not shown are several NOEs from

Val-197 and His-201 to the ethylene group of P1V. (From Ref. 6.)



structures were used to select structures for energy minimization using

the program FANTOM [49]. In the final calculations, 30 structures were

selected. Table 1 summarizes the DIANA and FANTOM statistics for

these structures.



VI. STRUCTURE OF INHIBITED STROMELYSIN-1

A. The Protein Fold

Superposition of residues 83 to 248 of the family of structures is shown

in Figure 9 viewed along the long axis of the catalytic helix. Residues 249

to 255 are disordered and therefore are not shown. In Figure 10 ribbon

drawings of two views of the molecule are shown, one from above the

h-sheet and the other from below the S1’ subsite. The secondary structure

of sfSTR consists of a five stranded h-sheet with four parallel strands and



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



Table 1 Structural Statistics and Residual Violations of the 30 Conformers Used

to Represent the Solution Structure of the Inhibited Catalytic Domain of

Stromelysin-1

Parameter



DIANA



DIANA target function (A˚ )

FANTOM energy (kcal/mol)

Lennard-Jones energy (kcal/mol)

Distance constraint violations (A˚)

sum

maximum

rmsd

Exp. angle constraint violations (j)

sum

Maximum

Rmsd

Rmsd residues 83 – 250 (A˚)

backbone (Ca,N,C’,O)

all heavy atoms

2



10.01 F 0.76



FANTOM

À191.0 F 52.8

À605.4 F 48.4



35.2 F 1.2

0.48 F 0.06

0.06 F 0.01



61.6 F 1.4

0.39 F 0.03

0.08 F 0.01



93.4 F 11.1

7.5 F 0.9

0.90 F 0.08



112.1 F 19.9

12.6 F 5.0

1.2 F 0.3



0.48 F 0.06

0.94 F 0.06



0.55 F 0.06

0.97 F 0.05



Source: Ref. 7.



one antiparallel strand and the topology À1x, +2x, +2, À1, using

the Richardson nomenclature [50]. The h-sheet lies on two helices (helix

A and B); a third helix (helix C) is near the C-terminus. The molecule

has two zincs: a catalytic zinc is located at the bottom of a cleft, and

a structural zinc above the h-sheet. The overall fold of sfSTR may

be described as follows. The N-terminus is located near the N-terminal

end of helix C. The protein backbone forms a poorly defined irregular

strand for the first 13 residues before entering strand I of the h-sheet,

then descending through helix A. Helix A acts as a backbone to the

protein, spanning its full length. The pronounced amphipaticity of this

helix provides hydrophobic residues for internal packing to helix B and

to the h-sheet, and the hydrophilic residues are exposed to the solvent.

After helix A the protein backbone turns to form strand II of the h-sheet,

which lies parallel to and outside strand I. This strand rises steeply, giving

the h-sheet a distinctly twisted appearance. It is connected by a short

loop to strand III, which is parallel to and inside of strand I. A long loop

connects strands III and IV, crossing over strand V and placing strand IV

along the ligand-binding cleft and antiparallel to strand V. Another small



Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved.



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IV. ASSIGNMENT OF THE RESONANCES OF THE INHIBITOR AND NOES BETWEEN THE PROTEIN AND THE INHIBITOR

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