Cell - 8 September 2016

The composition of the lumen is unique in being lined not only
with residues from thea2-a3 loop, but also with the lipid head
groups. In the F pilus, this loop, referred to as the ‘‘KNVK’’
loop, was hypothesized to form a contact with ssDNA during
conjugative transfer (Paiva et al., 1992). The structures pre-
sented here locate this loop to the lumen of the pilus, suggest-
ing that indeed the pilus serves as a conduit for ssDNA transfer.
Remarkably, integral to the lining of the lumen is the stoichio-
metric inclusion of phospholipid head groups. To gain further
insight into the potential impact that inclusion of PG head
groups within the lumen lining might have, the electrostatic
potential of the lumen was calculated with or without PG
(Figure 5C). Inclusion of PG has a profound impact on the elec-
trostatic potential of the pilus lumen: without PG, it is over-
whelmingly positive, while with PG, it is moderately electroneg-
ative. By generating a conduit with a moderately negative inner

surface, phospholipids may facilitate transport of the negatively

charged ssDNA substrate.

Mutational Studies of Lipid-Interacting Residues

Confirm the Importance of Lipid Binding in Preserving

the Integrity of the Pilus

The F TraA pilus subunit has been subjected to extensive muta-

genesis (Frost and Paranchych, 1988; Manchak et al., 2002). All

mutations observed to affect pilus biogenesis locate to protein-

protein interfaces, while mutations affecting conjugation and

phage attachment locate to either the lumen or the periphery

of the pilus. Thus, the structure presented here provides the

structural basis for all published F pilus mutations. However,

the PG-binding site was never targeted for mutation, as it was

unknown. Three residues in the interface between PG and

pED208 TraA were thus chosen for mutational analysis (the

A

B

Figure 4. MS Analysis of the Lipids Extracted from pED208 Pili
(A) Negative ion mode survey scan (600–780 mass/charge ratio [m/z]) of lipid extracts from whole-cell membranes.
(B) Negative ion mode survey scan (600–780 m/z) of lipid extracts from purified pili pre-treated with PLA2.
In all cases, phospholipids’ identity was confirmed by daughter fragmentation and reported here.

1440 Cell 166 , 1436–1444, September 8, 2016