Carboxisom

Structure

Article

Structure of the PduU Shell Protein
from the Pdu Microcompartment of Salmonella
Christopher S. Crowley,1 Michael R. Sawaya,2 Thomas A. Bobik,3 and Todd O. Yeates1,2,4,*
1University of California Los Angeles Molecular Biology Institute
2University of California Los Angeles-Department of Energy Institute for Genomics and Proteomics
University of California Los Angeles, Los Angeles, CA 90095, USA
3Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
4Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA

*Correspondence: yeates@mbi.ucla.edu
DOI 10.1016/j.str.2008.05.013

SUMMARY et al., 1998; Tsai et al., 2007). A few thousand copies of these
proteins are present in the shell of a single microcompartment.
The Pdu microcompartment is a proteinaceous, sub- In any given organism, these major shell proteins are typically en-
cellular structure that serves as an organelle for the coded in multiple homologous copies and arranged on the bac-
metabolism of 1,2-propanediol in Salmonella enter- terial chromosome near genes for metabolic enzymes, at least
ica. It encapsulates several related enzymes within some of which are either known or presumed to be encapsulated
a shell composed of a few thousand protein sub- by the shell. Some characterization has been performed on rep-
resentatives from three distinct metabolic classes of microcom-
units. Recent structural studies on the carboxysome,
partments, and up to seven distinct functional classes are pre-
a related microcompartment involved in CO2 fixation,
dicted based on genomic sequence analysis (Bobik, 2006). To
have concluded that the major shell proteins from date, detailed structural studies have been conducted only on
that microcompartment form hexamers that pack the carboxysome shell (Iancu et al., 2007; Kerfeld et al., 2005;
into layers comprising the facets of the shell. Here we Schmid et al., 2006; Tanaka et al., 2008; Tsai et al., 2007), a mi-
report the crystal structure of PduU, a protein from crocompartment that enhances carbon fixation in cyanobacteria
the Pdu microcompartment, representing the first and some chemoautotrophs by encapsulating essentially all of
structure of a shell protein from a noncarboxysome the ribulose-bisphosphate carboxylase-oxygenase (RuBisCO)
microcompartment. Though PduU is a hexamer like enzymes in the cell, together with carbonic anhydrase (Price
other characterized shell proteins, it has undergone a et al., 1992; Shively et al., 1973).
circular permutation leading to dramatic differences The pdu operon in Salmonella enterica serovar Typhimurium
LT2 (referred to hereafter as S. enterica) was recently determined
in the hexamer pore. In view of the hypothesis that
to encode proteins necessary for forming a microcompartment
microcompartment metabolites diffuse across the
for the B12-dependent metabolism of 1,2-propanediol (1,2-PD)
outer shell through these pores, the unique structure (Figure 1; Bobik et al., 1999; Chen et al., 1994; Havemann and
of PduU suggests the possibility of a special func- Bobik, 2003; Shively et al., 1998). The compound 1,2-PD is pro-
tional role. posed to serve as an important carbon and energy source for
aerobic and anaerobic metabolism in S. enterica and other bac-
INTRODUCTION terial species (Bobik, 2006). Several studies implicate the metab-
olism of 1,2-PD in bacterial pathogenesis (Adkins et al., 2006;
Bacterial microcompartments are large, virion-sized protein Conner et al., 1998; Heithoff et al., 1999; Klumpp and Fuchs,
assemblies comprised of specific enzymes encased within a 2007) and show that microcompartment formation is required
protein shell. They function as organelles by sequestering partic- for its efficient metabolic utilization as a sole carbon source (Ha-
ular metabolic processes within the cell. Microcompartments vemann et al., 2002; Sampson and Bobik, 2008).
have been observed directly by electron microscopy in several Havemann and Bobik (2003) recently characterized the total
species of bacteria (Figures 1C and 1D; Bobik et al., 1999; Brin- protein content of purified Pdu microcompartments from S. en-
smade et al., 2005; Drews and Niklowitz, 1956; Gantt and Conti, terica and determined that all but one of its 15 detectable pro-
1969; Jensen and Bowen, 1961; Shively et al., 1970), and their teins are encoded by the 21-gene pdu operon. Seven of these
relatively widespread existence across the eubacteria has proteins (PduA, -B, -B0 , -J, -K,-T, and –U) have a BMC domain.
been inferred by the presence of protein sequences homologous Immunogold labeling of microcompartments confirmed that
to the proteins that comprise the outer shell of diverse micro- PduA is present in the outer shell (Havemann et al., 2002); the
compartments (Bobik, 2006; Chen et al., 1994; Shively et al., other homologous BMC domain proteins (PduB, -B0 , -J, -K, -T,
1998; Stojiljkovic et al., 1995). In particular, the shells of varied and -U) are also presumed to be involved in formation of the
microcompartments are built primarily from small proteins be- shell. The Pdu microcompartments also incorporate four en-
longing to the bacterial microcompartment (BMC) domain family zymes involved in 1,2-PD metabolism (Figure 1B; Bobik et al.,
of proteins (Havemann et al., 2002; Kerfeld et al., 2005; Shively 1997, 1999; Johnson et al., 2001; Leal et al., 2003). The

1324 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved

Structure
Structure of a Pdu Shell Protein

High-resolution structural investigations of microcompart-
ment shell proteins have been initiated on the carboxysome (Ker-
feld et al., 2005; Tanaka et al., 2008; Tsai et al., 2007). Crystal
structures of four different BMC domain proteins showed that
they form cyclic hexamers with a tendency to pack into two-
dimensional layers representing the outer microcompartment
shell. In addition, all of the carboxysome shell proteins have a
˚
narrow pore, with a diameter in the 4–6 A range, running through
the center of the hexamer. These pores are predicted to allow
transport of the carboxysome substrates and products across
the protein shell (Kerfeld et al., 2005; Tsai et al., 2007).
It is likely that microcompartments of various types will follow
some of the same principles that have emerged from carboxy-
some studies, but with key differences accommodating their
unique metabolic functions. Pdu microcompartments are slightly
larger than typical carboxysomes and appear to be somewhat
less geometrically regular in shape (Figures 1C and 1D; Bobik
et al., 1999). In addition, compared with the carboxysome, the
Pdu microcompartment organizes different and more varied
enzymes in its interior, and must allow the diffusion of more
diverse metabolites and cofactors (Figure 1B; Bobik et al.,
1999). Structural studies on Pdu proteins are likely to shed light
on these elements of function.
We report here the crystal structure of the PduU shell protein
from S. enterica, the first BMC shell protein from a noncarboxy-
some microcompartment. PduU is one of the less abundant pro-
teins in the Pdu microcompartment (Havemann and Bobik, 2003)
and does not appear to be required for proper microcompart-
ment assembly, but its presence is required for optimal bio-
chemical function of the microcompartment (unpublished
Figure 1. The Pdu Microcompartment of Salmonella enterica data). We reveal here that PduU is a hexamer as expected, but
(A) Representation of the $17 kb pdu operon, whose genes are transcribed its three-dimensional topology reveals a surprising circularly per-
from a single promoter upstream from pduA (Bobik et al., 1992, 1997, 1999; muted variation on the typical BMC fold. A likely evolutionary
Chen et al., 1994). The pdu genes are labeled with their respective pdu suffix mechanism for the origin of the observed permutation is
designations, and drawn in proportion to their lengths and positions in the pdu presented. The resulting novel characteristics of the PduU
operon (Bobik et al., 1999). PduB and PduB0 arise from alternate start sites.
hexamer are discussed in light of their potential implications
Dark shaded arrows indicate genes whose encoded proteins are detected in
isolated microcompartments (Bobik et al., 1997, 1999; Havemann and Bobik,
for function in the Pdu shell.
2003; Havemann et al., 2002; Johnson et al., 2001; Leal et al., 2003). Addition-
ally, the genes marked by an asterisk (*) encode proteins homologous to the RESULTS
BMC shell proteins.
(B) A current model of the function of the Pdu microcompartment. The Pdu Structure and Assembly Features
microcompartment performs the initial steps in the metabolism of 1,2-pro- The crystal structure of PduU was determined by molecular re-
panediol (1,2-PD) to propionaldehyde (PA) and propionyl-CoA (PR-CoA) by ˚
placement and refined at a resolution of 1.8 A, with final R and
the B12–dependent diol dehydratase (PduCDE) and the aldehyde dehydroge-
nase (PduP), respectively (Bobik et al., 1997; Leal et al., 2003). The Pdu micro- Rfree values of 0.20 and 0.24 (see Experimental Procedures
compartment also contains the enzymatic reactions to generate (biologically and Table S1 available online). The crystal unit cell revealed
active) adenosyl-cobalamin (Ado-B12) from its precursor cob(I)alamin and to two cyclic hexamers in the R3 space group, each sitting on the
displace inactive deadenosylated B12 derivatives (represented by X-B12) threefold axis of symmetry. There are therefore four independent
from the diol dehydratase. These reactions are carried out by cob(I)alamin protein chains (A, B, C, and D), two from each hexamer, in the
adenosyltransferase (PduO) (Johnson et al., 2001, 2004) and diol dehydratase
asymmetric unit. The final atomic model is nearly complete; the
reactivase (PduGH), respectively (Bobik et al., 1999). Given this model, the
shell most likely allows passage of several relatively bulky substrates: 1,2-
first six residues were disordered in chains A and B from hex-
PD, PA, PR-CoA, and HS-CoA (coenzyme A), nicotinamide adenine amer 1, the first three residues were disordered in chain C, and
dinucleotide (NAD+) species, adenine nucleotides, and B12 derivatives. the first five residues were disordered in chain D from hexamer
(C and D) Transmission electron (TEM) micrographs of Pdu microcompart- 2. There were no significant discrepancies between the two in-
ments (arrows) within S. enterica (C) and of purified Pdu microcompartment dependent hexamers, and the protein backbones of chains A,
organelles (D). (Scale bar: 100 nm.) ˚
B, C, and D could be overlaid with an rmsd of <0.23 A. The qual-
ities of the models were checked using the program ERRAT (Co-
complexity of the Pdu microcompartment and its role in host lovos and Yeates, 1993) and Ramachandran plots; 94% of the
metabolic interactions make it an attractive target for structural residues were within the favored regions, and 99% of the resi-
and functional studies. dues were within the favored or allowed regions.

Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved 1325

Structure

Figure 2. Crystal Structure of the PduU
Shell Protein
(A and B) (A) The PduU hexamer viewed along
the sixfold axis and (B) perpendicular to the
sixfold axis with distinct protein chains colored
separately. The outline drawn around the
hexamer (A) illustrates its presumed packing
in the microcompartment shell among the
other (more abundant) homologous shell pro-
tein hexamers (e.g., PduA, PduB, PduB0 , and
PduJ). A prominent feature of the PduU hex-
amer is the six-stranded, parallel b-barrel
formed by one N-terminal b strand from each
monomer. Whether this feature at the top of
the hexamer faces out toward the cytosol or toward the interior of the microcompartment has not been established.
(C) Ribbon diagram depicting the PduU monomer, colored from blue (N terminal) to red (C terminal). Over residue positions 19–109, the chain adopts
a variation of the typical bacterial microcompartment (BMC) fold (Kerfeld et al., 2005). The 18 amino-terminal and eight carboxy-terminal residues
form novel secondary structural elements.

The PduU monomer adopts a compact, wedge-shaped fold ningham et al., 1979). This arrangement is unique among the
representing a variation on the previously described BMC fold carboxysome and other Pdu BMC proteins studied thus far.
(Figure 2C; Kerfeld et al., 2005). Six subunits form a cyclic hex- Circular permutation has been noted before as a mechanism
˚
amer roughly 70 A in diameter (Figures 2A and 2B). Within the for protein fold evolution (Grishin, 2001; Hahn et al., 1994; Pont-
hexamers, individual PduU subunits form extensive interactions ing and Russell, 1995). To examine the sequence similarity be-
with neighboring subunits, burying an average surface area of tween spatially analogous residues, a permuted version of the
˚
2626 A2 for each monomer. The natural hexameric state of PduU sequence was generated based on its three-dimensional
PduU was experimentally supported by native gel electrophore- alignment with the typical BMC core (Figures 3B and 3C; Fig-
sis experiments using a Ferguson plot (Ferguson, 1964) (data not ure 4). Sequence alignments between carboxysome shell pro-
shown). Though some of the basic features of the PduU hexamer teins of known structure and permuted PduU showed higher
match those reported for homologous carboxysome shell amino acid sequence identity than alignments with native
proteins, there are some dramatic differences in folding and PduU. The sequence identity in an alignment to carboxysome
assembly. shell protein CsoS1A, for example, increases to 21% for per-
Three of the four hexameric carboxysome shell proteins muted PduU, compared with only 14% for native PduU (see Fig-
whose structures have been determined (i.e., all but CcmK4) ure 4 for details). In addition, multiple sequence alignments
formed extended, two-dimensional molecular layers that are be- (Edgar, 2004) against diverse BMC protein sequences reveal
lieved to represent the outer shell of the microcompartment (Ker- the conservation of key residues that cannot be aligned using
feld et al., 2005; Tsai et al., 2007). In contrast, the two indepen- native PduU (Figure 4). For instance, using the permuted PduU
dent PduU hexamers observed in the present crystal are sequence, Ile26 and Pro29 (numbered as in the native PduU se-
arranged in loosely packed arrays (data not shown) that are quence) align with like residues in the C-terminal regions of other
probably not reﬂective of the arrangement of BMC protein hex- BMC proteins (e.g., Ile80 and Pro83 in CcmK4); using the native
amers in native Pdu shells. In one of the crystalline lattice layers PduU sequence ordering, those residues could not be aligned
(annotated as hexamer 1 in the crystal), the hexamers are touch- to conserved residues in the BMC domain (Figure 4). The
ing at their corners. In the hexamer 2 layer, which must be improvement in sequence identity, including key residues widely
spaced equally to the ﬁrst layer, the edges of neighboring conserved in BMC domain proteins, suggests that PduU is
hexamers are parallel, but the spacing is too large to permit inti- related to the typical BMC domain by an effective permutation
mate contact (contacts between hexamers in the crystal involve of the encoding gene sequence.
the histidine-tagged C-terminal tails). The transverse center- In addition to the permutation in the core, there are novel fea-
to-center distance between adjacent PduU hexamers in the tures in both termini of PduU, one of which is particularly signif-
˚ ˚
crystal is 74 A, compared with the range of 67–70 A for pre- icant. A minor elaboration is present at the C terminus, where
viously reported carboxysome hexamers in closely packed residues 109–116 contribute an extra strand to the edge of the
arrangements. core b sheet (Figure 3), whereas at the N terminus, an extension
in PduU creates an entirely new motif at the center of the hex-
amer. Owing to these variations, both ends of the protein chain
Structural Rearrangements in the BMC Fold terminate on the side of the hexamer opposite from that seen
The structure of PduU reveals a core that is similar to the typical in other BMC domain proteins.
BMC fold, but the sequential ordering of structurally aligned sec-
ondary structure elements is different (Figures 3B and 3C). The
secondary structure elements contributed by the C-terminal Central Pore
region of the typical BMC fold are instead contributed by the The N terminus of PduU extends toward the middle of the
N-terminal region of PduU. The relationship between the two hexamer, where residues 8–15 from each subunit contribute to
cores can therefore be described as a circular permutation (Cun- a parallel six-stranded b-barrel, which is unique among BMC


Structure

Figure 3. Structural Comparison of PduU
to Carboxysome Shell Proteins
(A–C) (A) Ribbon diagrams of superimposed pro-
tein backbones of PduU (yellow), CcmK4 (cyan),
and CsoS1A (magenta). The deviations were low
overall, but substantially elevated for PduU resi-
dues 76–79, which form a loop lining the central
cavity. Simplified diagrams of the arrangement of
secondary structure elements are shown for a typ-
ical BMC domain protein (B) and PduU (C). Addi-
tionally, the secondary structure elements are
labeled with respect to their occurrence in the
respective sequences. An apparent circular per-
mutation is evident.

domain proteins studied so far (Figure 5). Beginning at residue 8, from residues Met10, Gln12, and Tyr14 are oriented toward the b-
the barrel is comprised of the amino acid sequence DRMIQEYV. barrel interior. Beginning at the N-terminal opening of the barrel,
The b-barrel is broken by a subsequent proline residue at posi- the side chains from Met10 pack symmetrically about the sixfold
˚
tion 16. The barrel has an average diameter of $16 A, measured axis and nearly occlude the entire width of the b-barrel interior
between equivalent C-alpha positions on opposite sides of the (Figure 5). The six Gln12 side chains cannot be accommodated
˚
barrel, and a height of $12 A. The shear value, which describes in equivalent configurations inside the b-barrel because of steric
the degree of slanting of b strands in a barrel, is 12, which has collisions. Instead, the electron density supports modeling two
been noted to be a low-energy configuration for six-stranded side chain conformations, which alternate around the barrel
b-barrels (Murzin et al., 1994). A similar homo-oligomeric b-bar- (Figures 5A and 5B). In the ‘‘up’’ conformation, the side chains
rel, formed by seven b strands instead of six, is found in the are oriented toward the N-terminal pole of the b-barrel; in the
extramembrane domain of the heptameric mechanosensitive ‘‘down’’ conformation, the side chain amides are oriented to-
channel of small conductance (MscS) (Bass et al., 2002). Hereaf- ward the C-terminal pole. There are some minor conformational
ter we refer to the side of the PduU hexamer with the novel b-bar- differences in Gln12 packing between the two independent hex-
rel as the top; it is not known yet which side of the layer formed by amers visualized in the crystal. In one hexamer, down-oriented
assembled BMC proteins represents the inside versus the Gln12 side chains are oriented favorably for intermolecular hydro-
outside of the microcompartment. gen bonding between alternating side chain amide groups; in the
Although the central b-barrel in PduU sits on the axis believed other hexamer, up-oriented Gln12 side chains are oriented for hy-
to serve as a pore for transport in related BMC proteins, the drogen bonding between the amide groups (Figure 5A). The tight
barrel appears to be too tightly packed with large amino acid packing of methionine and glutamine side chains suggests that
side chains to allow efficient diffusion (Figure 5). The side chains efficient molecular transport down the center of the PduU

Figure 4. Sequence Comparison of Typical
BMC Domain Proteins to a Permuted
Version of PduU
The sequence alignment represents the four struc-
turally characterized BMC proteins. To perform
this alignment, a hypothetical PduU sequence
was constructed by permuting the native PduU
sequence according to a structural alignment be-
tween PduU and carboxysome shell proteins of
known structure (schematic, top). PduU sequence
segments were shuffled as follows (numbered ac-
cording to parent sequence position): residues
45–108, then residues 19–39. The ‘‘X’’ above
marks the position between adjoining segments.
Nonhomologous terminal sequence regions are
omitted here for clarity. The blue colored boxes
represent aligned, conserved residues, and the
corresponding PduU secondary structural ele-
ments are diagrammed above the alignment fol-
lowing the labeling presented in Figure 3C. The
PduU loop region containing residues D76–T79
(underlined with red) is shorter than the corre-
sponding loops in other BMC proteins of known
structure. Deviations between corresponding Ca
positions after superimposing CcmK2 and PduU
are graphed below in black bars (scale markers
in blue).


Structure

gous carboxysome shell proteins of known structure, and three
to ten additional residues in other Pdu BMC proteins (Figure 4).
The cavity on the bottom side of the PduU hexamer appears to
present an unusually hydrophobic surface. A high percentage of
the surface of the cavity is comprised of the exposed aromatic
faces of side chains from Tyr14 and Phe78, which contribute an
˚ ˚
average of 74 A2 and 125 A2 per subunit, respectively (Figure 6B).
The Tyr14 residues at the C-terminal end of the central b-barrel
protrude from the barrel in a ring, with the aromatic plane nor-
mals oriented nearly perpendicular to the central axis of symme-
try. The aromatic faces of Phe78 side chains are arranged simi-
larly in a ring near the bottom opening of the cavity. Sequence
alignments with other Pdu proteins suggest that this aromatic
surface is unique to PduU. Less substantial, but significant, con-
tributions to the surface are made by Ser52, Thr54, and Glu55;
˚ ˚ ˚
these contribute an average of 12 A2, 17 A2, and 7 A2 per subunit,
respectively (Figure 6B). The relatively hydrophobic character of
this surface suggests the likelihood of native interactions with
other molecules—either other proteins or smaller molecules—
that have not yet been visualized.

DISCUSSION

The structure of PduU recapitulates some of the features that
have been observed in structures of the major shell proteins of
the carboxysome; the core of PduU is related to the typical
BMC domain, and its natural oligomeric state is a cyclic hexamer
Figure 5. Structure of the b-barrel Cap in PduU (Kerfeld et al., 2005; Tsai et al., 2007). Beyond this, however,
(A and B) Cutaway views showing the side chain packing within the two hex- PduU reveals unique features that are likely to relate to its func-
amer b-barrels. The side chains (sticks) of Met10, Gln12, and Tyr14 are buried
tion. At least three functional properties would seem to be impor-
within the b-barrel. Owing to steric restrictions, all Gln12 side chains were mod-
eled in two alternating conformations: ‘‘up’’ toward the N terminus and ‘‘down’’
tant for the major shell proteins of bacterial microcompartments.
toward the C terminus. Interchain hydrogen bonding between upward-oriented The hexameric proteins must interact with each other to form
Gln12 side chains is present in the configuration shown in (A) (dashed line). a layer that surrounds the organelle. Additionally, they must
(C) Stereo view of the PduU b-barrel viewed from its C-terminal end. Gln12 and make critical interactions with the interior components of the mi-
other interior side chains are illustrated. The configurations shown in both crocompartment, and allow movement of small molecules
panels are from the hexamer labeled ‘‘1’’ in the crystal asymmetric unit.
across the shell. The structure of PduU provides functional in-
sights into each of these issues.
By itself, PduU does not appear to form well-packed hexago-
hexamer would not be possible, at least not with the N terminus nal layers as easily as the carboxysome proteins that represent
in the configuration observed here. We note that, notwithstand- major constituents of that shell. This property is shared by
ing the minor differences in amino acid side chain orientations, PduU and one particular carboxysome shell protein, CcmK4
the N terminus of PduU adopts nearly equivalent configurations (Heinhorst et al., 2006; Kerfeld et al., 2005). Neither PduU nor
in the two independent hexamers in the crystal, arguing against CcmK4 is a major constituent of its respective shell (Havemann
the b-barrel being a spurious structure. et al., 2002; Heinhorst et al., 2006). The low abundance of these
The opposite side of the PduU hexamer is likewise unique. proteins is consistent with their failure to form layers on their
Whereas the top side is blocked by the N-terminal b-barrel, the own, since they presumably interact with homologous but dis-
bottom side of the PduU hexamer is opened much wider at the tinct hexamers that are more abundant in native shells. The
site of the central pore than other structurally characterized need for multiple homologs of the BMC domain proteins—and
BMC domain proteins (i.e., those from the carboxysome; Fig- this is the case for every genome in which they have been
ure 6). Viewed in cross-section with the central b-barrel at the found—is not fully understood. In the case of CcmK4, the ten-
top, the result is a roughly bell-shaped cavity or deep depres- dency of that protein to form strips instead of layers has led to
sion, with Gln12, which is near the C-terminal end of the b-barrel, the speculative suggestion that it could occur at edges of the
forming the apex. Whereas the analogous central pores of the icosahedral shell (Kerfeld et al., 2005). In the case of PduU, the
˚
carboxysome hexamers are narrow (4–7 A in diameter), the cen- inability to form homogeneous PduU hexamer layers might be
tral cavity on the bottom side of the PduU hexamer is approxi- a mechanism for limiting its incorporation into Pdu microcom-
˚
mately 17 A across (Figure 6B). The widened opening in PduU partment shells.
is due mainly to a loop truncation, rather than differences in One of the major structural differences seen in PduU is the
amino acid side chains. In PduU, the loop between residues 76 deep cavity or depression on one side of the hexamer (Figures
and 79 replaces a loop that has three more residues in homolo- 6A and 6B). This open cavity takes the place of a much narrower


Structure

pore observed at the hexameric axis in the BMC domain proteins
from the carboxysome shell (Figures 6A and 6C). The surface of
the cavity in PduU presents several hydrophobic residues
(Figure 6B), which suggests the likelihood of yet unknown molec-
ular interactions. If the purpose of the hydrophobic surface is to
establish speciﬁc interactions with interior components of the
microcompartment, then that side of the hexameric layer would
have to face the microcompartment interior. Which side of the
hexameric layer of BMC domain proteins represents the outside
of a microcompartment and which side faces inward is a ques-
tion that has not been resolved for the carboxysome or the
Pdu microcompartment.
Another unique feature in PduU is the intermolecular six-
stranded b-barrel that appears to block the central pore that ex-
ists in other BMC domain proteins (Figure 4). Beta-barrels occur
often in transmembrane proteins, where they create pores for
molecular transport (Schulz, 2002). However, b-barrels in struc-
turally characterized transport proteins are comprised of at least
12 strands, making them considerably larger in diameter than the
PduU b-barrel. Given its narrow diameter, one hypothesis is that
instead of serving a transport function, PduU might serve to orga-
nize a particular enzymatic component in the interior of the micro-
compartment. An alternative hypothesis is that the b-barrel in
PduU could open up under certain conditions to allow transport.
The b-barrel in PduU is similar in certain respects to the C-termi-
nal, symmetric seven-stranded b-barrel from the MscS mecha-
nosensitive channel (Bass et al., 2002), which has likewise been
proposed to undergo structural transitions (Koprowski and Ku-
balski, 2003). If the b-barrel in PduU is capable of opening, then
the unusually wide entrance on the other side of the PduU hex-
amer (discussed previously) would allow the diffusion of larger
molecules than those thought to pass through the pores of
corresponding carboxysome shell proteins. Although it is not en-
tirely clear which compounds must cross into and out of the Pdu
microcompartment, some of the bulkier substrates might include
nucleotides, nicotinamide adenine dinucleotides, coenzyme A
species, and coenzyme B12 derivatives (Figure 1B; Bobik,
2006; Chen et al., 1994). The thermal displacement parameters
(B-factors) were examined in the region of the b-barrel, but
were not found to be signiﬁcantly elevated compared with the
rest of the protein. Further studies will be required to test hypoth-
eses about the potential roles of PduU in binding and transport.
Some of the key structural differences between PduU and
other BMC domain proteins arise from the apparent circular per-
mutation in PduU (Figures 3 and 4). What evolutionary sequence
of events might have led to this genetic rearrangement? The ap-
parent circular permutation could have arisen by tandem gene
duplications of a more typical BMC-type protein in the PduU
operon, followed by truncation at the ends of the duplicated gene
Figure 6. Properties of the Central Cavity in the PduU Hexamer
to yield a permuted single domain. This scenario is consistent
(A) Comparison of a carboxysome shell protein hexamer, CcmK4 (orange)
(Kerfeld et al., 2005), with the PduU hexamer (blue). To illustrate differences from carboxysome shell proteins whose pores are lined mainly by charged and
at the PduU central pore region, the N termini of PduU are truncated so that polar side chains (Kerfeld et al., 2005; Yeates et al., 2007).
only residues 19–116 are shown. (C) A corresponding view of CcmK4, a carboxysome shell protein hexamer.
(B) A cutaway view of the central cavity of the PduU hexamer, which is ˚
Measurement bars are added for scale, representing approximately 17 A (B)
suggested here to be oriented with the open cavity facing the lumen of the ˚
and 8 A (C). They indicate the distance between the Cb atoms of Phe78 from
Pdu microcompartment. The image is colored by atom type, with carbon opposing protein chains in (B) and between Arg38 N3 side chain atoms from
(green), nitrogen (blue), and oxygen (red). Side chains from residues Met10, ˚ ˚
opposing chains in (C) (17.1 A and 7.8 A, respectively). Note that the pore
Gln12, Tyr14, and Phe78 are shown to illustrate their contributions to the surface. that is present in the carboxysome shell protein hexamer (C) is blocked by
The substantial surface contributions by aromatic side chains are a distinction the b-barrel in PduU (B).


Structure

Figure 7. Cartoon Representations of the
Seven S. enterica Pdu Shell Proteins Con-
taining BMC Domains Recognizable by Se-
quence Homology
The boxes outline the sequence segments con-
taining predicted BMC domains. The typical
BMC domains are shaded from white at the N
terminus to black at the C terminus. PduU can be
aligned to parts of two consecutive BMC domains,
or can alternatively be described as a circular
permutation of a single BMC domain.

with a model first described in the saposin family (Ponting and sion method. The protein crystallized in 25 of 480 initial screening conditions, in
Russell, 1995) and is supported by the frequent appearance of most cases forming crystals with hexagonal plate morphologies. Crystal con-
ditions typically contained 20% (v/v) low molecular-weight polyethylene glycol
multiple BMC protein genes encoded adjacent to each other
(PEG), overall ionic strength <500 mM, and pH range 6.0–8.0. An optimized
(Bobik et al., 1999), as well as the existence of individual micro- condition with 50 mM HEPES (pH 8.0), 300 mM LiSO4, and 20% PEG-3350
compartment shell proteins apparently containing two tandem produced good single crystals with the morphology of hexagonal plates.
BMC domains. PduB and PduT illustrate two such cases Single crystals were harvested in nylon loops, cryoprotected by the
(Figure 7). addition of 30% (v/v) ethylene glycol, and then frozen for subsequent data
As a model system for studying bacterial microcompart- collection in a nitrogen stream at À176 C. A full data set was collected to
˚
a resolution limit of 1.8 A at the Advanced Light Source Beamline 8.2.2
ments, Salmonella offers two key features. Protocols have
(Berkeley, CA; Table S1).
been developed for isolating Pdu microcompartments in intact
form (Havemann and Bobik, 2003), and genetic manipulation Structure Determination and Analysis
in Salmonella is straightforward. This opens up the possibility Diffraction data were indexed and processed using the programs DENZO and
of isolating mutant microcompartments for in vitro experiments; SCALEPACK (Otwinowski and Minor, 1997). The program PHASER (McCoy
this has not yet been possible for carboxysomes, which are et al., 2005) was used to solve the structure by molecular replacement using
difficult to purify from bacterial species that are amenable to as a search model the crystal structure of CsoS1A, a hexameric carboxysome
shell protein from H. neapolitanus (Protein Data Bank accession code 2EWH;
genetic manipulation (Heinhorst et al., 2006). S. enterica also
Tsai et al., 2007). The model was built using the graphics program COOT
produces a so-called Eut microcompartment for the metabo- (Emsley and Cowtan, 2004) and refined using the program REFMAC (CCP4,
lism of ethanolamine, which might be amenable as well to 1994; Murshudov et al., 1997). NCS restraints were applied at a medium
combined biochemical and genetic studies (Stojiljkovic et al., strength setting to the four molecules in the asymmetric unit. Restraints
1995). Structure-guided mutagenesis in the Pdu and Eut sys- were released for the last five rounds of the refinement. The final R and Rfree
tems should make it possible to answer unresolved questions values were 20.3% and 24.2%, respectively (Table S1).
The Pymol molecular graphics program (http://www.pymol.org/) was used
about bacterial microcompartments.
for visual investigation, molecular measurements, and generation of cartoon
structure images of the PduU structure. Measurement bars and labels were
EXPERIMENTAL PROCEDURES added to exported images in the image-rendering program Photoshop
(Adobe Systems; San Jose, CA). Surface area measurements were calcu-
Transmission Electron Microscopy lated by the program AREAIMOL (Lee and Richards, 1971). Superpositioning
Transmission electron microscopy imaging of Salmonella enterica serovar of Ca backbone atoms was performed by the program SUPERPOSE (Krissi-
Typhimurium LT2 sections and purified Pdu microcompartments was per- nel and Henrick, 2004). Root mean squared deviations between pairs of
formed as previously described (Bobik et al., 1999; Havemann and Bobik, structures were calculated over the following chain segments: CcmK2
2003) using a Zeiss EM-10CA transmission electron microscope (Zeiss; Jena, residues 71–90, CcmK4 residues 73–92, and CsoS1A residues 74–93 over
Germany). PduU residues 19–38; CcmK2 residues 4–37, CcmK4 residues 6–39, and
CsoS1A residues 8–41 over PduU residues 45–78; and CcmK2 residues
Cloning and Protein Purification 41–69, CcmK4 residues 43–71, and CsoS1A residues 45–73 over PduU
Briefly, the gene pduU from S. enterica was amplified from the plasmid con- residues 79–107.
struct MGS2 (Bobik et al., 1999) for cloning into the plasmid pET22b (Novagen;
Darmstadt, Germany). The resulting construct encodes full-length PduU (116 ACCESSION NUMBERS
amino acids) plus a C-terminal Leu-Glu-His6 affinity sequence. Protein expres-
sion from sequence-verified PduU-pET22b constructs (hereafter PduU) was Coordinates have been deposited in the Protein Data Bank with the accession
carried out in BL21 (DE3) RIL+ E. coli cells (Stratagene; La Jolla, CA) in 1 liter code 3CGI.
cultures of Luria-Bertani (LB) media + 1% glucose. Protein was purified from
the soluble cell fraction by HisTrap HP affinity chromatography (GE Health- SUPPLEMENTAL DATA
care; Waukesha, WI) followed by Q Sepharose HP anion-exchange chroma-
tography (GE Healthcare). All steps were carried out in the presence of Supplemental Data include one table and Supplemental References and can
0.2 mM PMSF and 1 mM DTT. Twelve percent SDS-PAGE was performed be found with this article online at http://www.structure.org/cgi/content/full/
to monitor the purity of the sample. 16/9/1324/DC1/.

Crystallization and Data Collection ACKNOWLEDGMENTS
Protein for crystallization was concentrated to 9 mg/ml in 25 mM Tris (pH 8.0)
and 25 mM NaCl. A Mosquito robotic instrument (TTP Labtech; Melbourn, The authors thank the staff at ALS synchrotron beamline 8.2.2 for technical
U.K.) was used to set up crystallization trials by the hanging drop vapor diffu- assistance; Duilio Cascio for crystallographic advice; Morgan Beeby for gel


Structure

electrophoresis analysis; James Stroud, Christopher Miller, Shiho Tanaka, and Grishin, N.V. (2001). Fold change in evolution of protein structures. J. Struct.
Yingssu Tsai for helpful comments; and Cheryl Kerfeld for a critical reading of Biol. 134, 167–185.
the manuscript. This work was supported primarily by the Office of Biological Hahn, M., Piotukh, K., Borriss, R., and Heinemann, U. (1994). Native-like in vivo
and Environmental Research of the U.S. Department of Energy, Office of Sci- folding of a circularly permuted jellyroll protein shown by crystal structure
ence (T.O.Y.) and by a grant from the National Science Foundation analysis. Proc. Natl. Acad. Sci. U.S.A. 91, 10417–10421.
(MCB0616008 to T.A.B.).
Havemann, G.D., and Bobik, T.A. (2003). Protein content of polyhedral organ-
elles involved in coenzyme B12-dependent degradation of 1,2-propanediol in
Received: March 20, 2008
Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 185, 5086–5095.
Revised: May 29, 2008
Accepted: May 30, 2008 Havemann, G.D., Sampson, E.M., and Bobik, T.A. (2002). PduA is a shell pro-
Published: September 9, 2008 tein of polyhedral organelles involved in coenzyme B(12)-dependent degrada-
tion of 1,2-propanediol in Salmonella enterica serovar Typhimurium LT2. J.
Bacteriol. 184, 1253–1261.
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