Supplementary Information for Sukharev et al.
Description of the Molecular Modeling Process and Rationale
In general, models of all conformations were developed to be consistent with the experimental data and to
satisfy a series of modeling criteria 17 related to (1) energetically favorable interactions of residues with
water, lipids, and each other; (2) favorable backbone and side chain conformations; (3) clustering of highly
conserved residues and exposure of poorly conserved residues along the series of conformations
representing the transition 18; (4) helix packing theory 11,19 ; and (5) the thickness of a lipid bilayer and
formation of a tightly packed protein barrier between membrane lipids and water of the pore.
Specifically, the following procedures were used to develop the structural models. First, models of the
helical portions of EcoMscL were developed using a program that generates  helices with idealized
backbone conformations and side chains in the conformation that occurs for that residue most frequently in
 helices. The M1 and M2 helices were then matched to those of the TbMscL crystal structure. The S1
helices and S3 helices were arranged to generate two different bundles of helices with 5-fold symmetry that
each have a highly conserved hydrophobic core and in which helices packing is consistent with ‘knobsinto-holes’ and or ‘ridges-into-grooves’ helix packing theories. The initial models of the S1 and S3 bundles
were left-handed, which allows the helices to pack in a manner typical of coiled-coils. These were placed
in the cytoplasmic region with the S3 bundle located as it is in the TbMscL crystal structure and with the
S1 bundle positioned co-axially between the S3 and M1 bundles. Segments connecting the helices were
then constructed manually choosing backbone and side chain conformations that are energetically
favorable. The axial location and radial orientation of the S1and S3 bundles were adjusted to enhance
energetically favorable interactions and conformations of the linking segments. The periplasmic loop of
EcoMscL was so unlike that of TbMscL that it was generated strictly to satisfy our criteria, using an  helix
for the initial region and a distorted  hairpin for the remaining portion. Some side chain conformations
were then adjusted to alternative conformations that are energetically favorable to eliminate energetically
unfavorable contacts and to enhance energetically favorable interactions such as salt bridges and hydrogen
bonds. The energy of the model was then minimized using CHARMM with the structure constrained to
maintain five-fold symmetry about the pore’s axis. The minimized model was then examined visually and
adjustments were made manually to make the models more consistent with our criteria. The adjusted model
was then minimized again. This process was repeated until no additional improvements could be made.
Two classes of models have been developed for the open conformation, 10-helix and 5-helix pore models.
The most obvious mechanism for opening the pore is one in which the M1 helices move radially outward
between the M2 helices, creating an open pore lined by both M1 and M2 helices (10-helix pore) 5,6,9. We
developed several models of this type. None of these models satisfied our criteria as well as the 5-helix
pore models presented here. Specifically, more hydrophobic side chains, primarily on M2, were exposed to
water inside the pore and there were fewer energetically favorable contacts among highly conserved
residues.
In the framework of 5-helix pore model, the opening of the channel is a result of a gradual expansion of the
barrel associated with increased tilts of transmembrane helices. First, the tightly packed M1 and M2 pair
from adjacent subunits was translated radially outward about one Å and the tilts and orientations of this pair
were adjusted to eliminate gaps between the helices, to minimize the amount of hydrophobic surface that is
exposed to water inside the channel, and to maintain ‘4-4 ridges-into-grooves’ packing between adjacent
M1 helices. The locations and orientations of the S1 and S3 bundles and conformations of all of the
connecting segments were constructed in a manner that minimized changes from the closed conformation
and that maintained a tight seal between the S1 bundle and the cytoplasmic entrance to the transmembrane
pore. The initial, slightly expanded, structure was then minimized using CHARMM. The process of
iterative manual adjustments of this model followed by energy minimization was repeated as before. The
process of expanding the transmembrane pore structure in small steps and optimizing the structure was
repeated in successive steps until, when the M1 pore had a diameter of 10-15 Å, the increase of the tilts of
M1 and M2 reduced the transmembrane distance spanned substantially, the exposed hydrophobic surface
on M1 and M2 began to increase substantially, opening began to form between the S1 bundle and the M1M2 pore, and the M2-S3 linker became very extended. The amphipathic S3 helices were then placed
parallel to the membrane at its cytoplasmic surface in a location where they blocked the openings between
the S1 bundles and the pore. In these expanded-closed models the highly conserved hydrophobic residues
of the S3 helices interact with lipids and/or with other highly conserved hydrophobic residues on M1 and
M2, while their highly conserved charged residues form salt bridges with other highly conserved charged
residues of the S1 helices. The process of gradually expanding the transmembrane pore while maintaining
the tightly packed S1 bundle was then continued until its diameter was ~25 Å and the R13-G14-N15
segment linking S1 to M1 was fully extend. The S1 bundle was then adjusted to form a right handed
bundle similar to that formed by the M1’s of the closed conformation. The hydrophobic core at the axis of
this bundle is formed only by the F7 and I3 side chains. The F10 side chain is more peripheral; it fits
between the I3, I4, F7, and R8 side chains of the adjacent S1 helix (see Fig. 3). The right-handed S1
bundle was further stabilized by the binding of the highly conserved E6 side chain to the N-terminus of the
adjacent S1. The C-terminus of the S1’s in the right-handed bundles is farther from the axis of symmetry
than in the left-handed bundles, which reduced the tension on the S1-M1 linker and allowed additional
expansion of the transmembrane pore prior to disruption of the S1 bundle. When the pore reached a
diameter of about ~30 Å, the S1-M1 linker became fully extended even when the S1 helices are maximally
tilted for the right-handed bundle. This was judged to be the closed conformation with the largest possible
transmembrane pore. Models were then developed of open conformations in which S1 helices dock on the
parameter of the channel. These models were developed so that the highly conserved F7 and F10 side
chains of S1’s could interact with highly conserved side chains of I25, A28, and F29 of M1 and F85, A89,
and F93 of M2. This docking displaces the S3 helix from its previous site. S3 was repositioned parallel to
the S1 helix so that its hydrophobic residues should interact with lipid alkyl chains at the cytoplasmic
surface while it’s highly conserved charged residues still form salt bridges with highly conserved charged
residues of S1. This type of model was expanded a bit more until the pore’s diameter of was ~38Å, which
we deemed to be the largest pore that could be developed realistically with this type of model. This size is
consistent with experimental findings 7,8.
Also, during the expansion process, portions of the S2 periplasmic loop were gradually pulled into the
transmembrane region. The loop was constructed so that most of its hydrophobic residues are buried when
the channel is closed but become exposed to lipid alkyl chains on outer surface as the channel expands.
In all, we developed models with transmembrane pores having thirteen different sizes, ranging from fully
closed as in the crystal structure to fully open with a diameter of 38 Å.
The transitions involved little changes in secondary structures with most of the conformational changes
occurring at or near glycines or prolines (G14 of the S1-M1 linker; P43 and P44 between the M1 and S3
helix; G50 and G51 between the S3 helix and first S3 β strand; G66 and P69 in the β turn region of S3; G76
between S3 and M2; and P109, P113, and P115 of the linker between the M2 and S3 helices). Glycines
and prolines tend to introduce flexibility into protein structures. With the exceptions of the break down of
the S1 and S3 bundles and docking of the S1 and S3 helices on the perimeter of the channel, all of the
conformational changes between successive steps are relatively small with no apparent steric energy
barriers for the transitions from one step to the next. In all conformations, almost all of the hydrophobic
side chains are either buried, partially buried, or located where they should be exposed to lipid alkyl chains.
In the open conformation hydrophobic side chains of M1, V23, F29, V33, V37, and I41, are partially
exposed to water in the pore, which should contribute to the instability of the open conformation when the
membrane is not stretched. All of the hydrophilic side chain atoms are exposed to water and/or form
energetically favorable salt bridges or hydrogen bonds in all conformations. The M1 helices have a series
of small ambivalent glycine, alanine, and serine residues that are buried in the closed conformation and
become exposed in the expanded conformations. The energy change in moving these residues from a
buried protein environment to an exposed aqueous environment is small. The barrier between the
hydrophobic lipid phase and interior of the pore is solid in all conformations and the length of the
hydrophobic surface on the exterior of the model is sufficient to span a hydrophobic region that is ~30 Å
thick. Sequences of thirty five MscL homologs were aligned and the degree of conservation at each
position of alignment was determined. With the exception of some glycine and proline residues that affect
the backbone structure, all highly conserved residues interact with other highly conserved residues in at
least one conformation, and most interact with other highly conserved residues in all conformation. These
highly conserved interactions are energetically favorable; e.g., all charged side chains form salt bridges in
at least one conformation, all hydrophobic side chains interact with other hydrophobic side chains, and
aromatic residues tend to form clusters.