C O M M U N I C A T I O N S
Table 2. Siderocalin Affinity for Ferric Ent and Salmochelin S4
Analogues at pH 7.4
siderocalin ligand
Kd, nM (esd’s)
[FeIII(SERMECAM)]3-
no binding
no binding
0.32(1)
0.49(6)
1.93(70)
[FeIII(SER-t-BUCAM)]3-
[FeIII(TRENCAM)]3-
[FeIII(TREN(CAM)2(MECAM)]3-
[FeIII(TREN(CAM)(MECAM)2]3-
not interfere with the conformation of the protein calyx, insertion
of a third methyl group, as on SERMECAM, hinders siderocalin
binding.
Figure 3. Fluorescence quenching titrations to measure the affinity of
siderocalin (100 nM) for [FeIII(TRENCAM)]3- and [FeIII(SER-t-BUCAM)]3-
Symbols give the fluorescence measurements at 340 nm, and lines give
the calculated fits. (Insert) Siderocalin cocrystallized in the presence of
[FeIII(SER-t-BUCAM)]3-(top) and [FeIII(TRENCAM)]3- (bottom).
.
While our results demonstrate that electrostatics contribute
extensively to the overall affinity of siderocalin for [FeIII(Ent)]3-
,
cation-π interactions likely dominate. Gaps in the siderocalin-
[FeIII(Ent)]3- adduct structure suggest positions on the siderophore
where modifications could be accommodated.13 The siderocalin
calyx tolerates considerable variation of the binding siderophores,
such as replacement of the Ent trilactone with the TREN scaffold,
while simultaneously imposing significant steric constraints, such
as not tolerating methylation of the three catecholate rings. Our
data support the idea that Ent glucosylation may, in part, be a
bacterial response to evade siderocalin-mediated iron depletion.
To further probe the steric constraints imposed by the rigid calyx
of the protein, two analogues of enterobactin and salmochelin S4
were synthesized: SERMECAM and SER-t-BUCAM. These
incorporate the same serine trilactone backbone of the natural
siderophores but also contain bulky alkyl (methyl and tert-butyl,
respectively) substituents on the 5′ position of each catecholamide
unit (Figure 1). Coupling of the trilactone trihydrochloride salt to
the methyl- or tert-butyl-substituted benzyl-protected catechol units,
followed by catalytic hydrogenation, afforded the new ligands in
good yields (Scheme S1). The affinity of siderocalin for the ferric
complexes of these two synthetic analogues was subsequently
investigated. The intrinsic fluorescence of the system was not
significantly quenched upon addition of [FeIII(SERMECAM)]3- or
[FeIII(SER-t-BUCAM)]3- (Figures 3 and S2). Cocrystallization of
siderocalin in the presence of each ferric complex also confirmed
no binding of these compounds by the protein. Colorless crystals
were obtained in both cases (Figures 3 and S2), indicative of the
absence of the ferric-catecholate complexes in the calyx of the
protein, since such ferric complexes exhibit a characteristic deep-
red color resulting from the intense ligand-to-metal charge-transfer
transitions.13,14 Addition of bulky alkyl groups on all catecholate
units of Ent therefore introduces sufficient steric hindrance to
preclude binding to siderocalin.
In an attempt to determine the maximal size and shape of the
siderophore that can be bound by the protein, another series of
analogues was designed: TRENCAM,16 TREN(CAM)2(MECAM),
and TREN(CAM)(MECAM)2. These form a sequence of tripodal
ligands, with a systematic addition of methyl groups on the 5′
position of the aromatic rings (Figure 1). The TREN scaffold was
used (instead of the triserine cyclic frame) to permit slow addition
of thiazolide-activated catecholamide units, and thereby control the
stoichiometry of the substituted moieties (Scheme S2). The affinity
of siderocalin for [FeIII(TRENCAM)]3- was measured by fluores-
cence titrations (Figure 3). The similar equilibrium dissociation
constants obtained for the ferric complexes of TRENCAM and Ent
(Table 2) demonstrate that replacing the triserine backbone by the
tripodal TREN scaffold does not affect siderocalin recognition.
Therefore TREN(CAM)2(MECAM) and TREN(CAM)(MECAM)2
are relevant intermediates between Ent and the bulky SERMECAM.
Each insertion of a methyl group on the catecholate rings of the
tripodal ligands resulted in a slight decrease of the affinity of
siderocalin, as indicated by the increase of the measured Kd values
(Table 2). Although the structure of TREN(CAM)(MECAM)2 does
Acknowledgment. This work was supported by the National
Institutes of Health (K.N.R.: AI11744, R.K.S.: AI59432). We
thank Anh Phan for help with the synthesis, Wendy Paulsene for
help with the protein, and Trisha Hoette for useful discussions.
Supporting Information Available: Detailed experimental pro-
cedures for ligand synthesis, fluorescence titrations, and cocrystallization
assay; synthetic schemes and additional titration data. This material is
References
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