Siderocalin Combats Mycobacterial Infections - Iron Acquisition by the Genus Mycobacterium - page 58

Although the Scn calyx seems optimized for binding three aromatic rings
in the three binding pockets lined with positive residues, the protein can
also accommodate siderophores and iron-siderophore complexes of varying
structures, so long as steric and electrostatic/cation-pi requirements are met.
So-called stealth siderophores are structurally modified such that these and
the ferric complexes preclude binding by Scn due to clashes with Scn bind-
ing pocket walls. Examples are the anthrax pathogen siderophore, petrobactin,
which has 3,4-catecholate iron binding moieties instead of 2,3-catecholates, and
salmochelins, which are glycosylated enterobactin structures. Furthermore, Scn
does not usually bind hydroxamate-based siderophores or corresponding iron
complexes since these substrates lack the aromatic electronic structure necessary
for cation-pi interactions with the protein [ 14 ]. On the contrary, siderophores
with structures that vary drastically from enterobactin can still be accommodated
by Scn. The carboxymycobactins are one example; although these siderophores
bind iron with two hydroxamate units and one hydroxyphenyl-oxazoline moi-
ety, some ferric carboxymycobactins are sequestered by Scn, as will be further
explained.
Aside from the aforementioned iron-binding moieties, carboxymycobactins
also have a fatty acid tail of varying lengths (Fig. 4.2 , n = 3-10) which has some
impact on Scn affinity for the corresponding ferric complexes [ 10 ]. The affini-
ties of ferric carboxymycobactin structures for Scn have been evaluated in vitro
with fluorescence quenching binding assays to determine the protein-ligand dis-
sociation constants ( K D ) as well as with protein crystallography to obtain an accu-
rate, three-dimensional input as to why certain isoforms are better ligands than
others. Hoette et al. [ 10 ] (Table 4.1 ) have shown that Scn has the greatest affin-
ity for carboxymycobactin isoform of n = 7, 8 and cannot sequester isoforms as
well for n = 3-6. The K D for the n = 7 carboxymycobactin isoform was the low-
est (128 nM), while some others had much higher K D 's (e.g. >900 nM for n = 3)
essentially indicating no significant affinity.
Crystal structures of ferric carboxymycobactin bound by Scn have provided a
rationale for the binding trend. Ferric carboxymycobactin structures for n = 5, 6,
7 were crystallized within the Scn calyx. Common to all structures, the hydroxy-
phenyl-oxazoline of all carboxymycobactins resides in pocket 1 and serves as the
main site of Scn affinity due to it being the sole aromatic moiety in carboxymy-
cobactins. The protein structures co-crystallized with ferric carboxymycobactins
also revealed that the fatty acid chain length of carboxymycobactins adopts either
a 'tail-in' or 'tail-out' (Fig. 4.3 ) conformation within pocket 2. This conformation
change has the most drastic effect on Scn affinity. The carboxylate tail, when in
'tail-in' conformation, creates a crucial interaction between calyx and ligand and
thus helps carboxymycobactins reside in the Scn calyx with higher affinity. By this
same token, carboxymycobactin fatty acid tails which are too short (n = 3) cannot
maintain this interaction with the binding pocket, thus resulting in less favorable
binding to Scn. The structure of Scn: carboxymycobactin (n = 5) has previously
been solved with the carboxymycobactin tail being both tail-in and tail-out in dif-
ferent crystal structures (Fig. 4.3 ) [ 10 ].