Uploaded on


More in: Technology
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Be the first to comment
    Be the first to like this
No Downloads


Total Views
On Slideshare
From Embeds
Number of Embeds



Embeds 0

No embeds

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

    No notes for slide


  • 1. Structure ArticleStructure of the PduU Shell Proteinfrom the Pdu Microcompartment of SalmonellaChristopher S. Crowley,1 Michael R. Sawaya,2 Thomas A. Bobik,3 and Todd O. Yeates1,2,4,*1University of California Los Angeles Molecular Biology Institute2University of California Los Angeles-Department of Energy Institute for Genomics and ProteomicsUniversity of California Los Angeles, Los Angeles, CA 90095, USA3Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA4Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA*Correspondence: yeates@mbi.ucla.eduDOI 10.1016/j.str.2008.05.013SUMMARY 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 leastica. It encapsulates several related enzymes within some of which are either known or presumed to be encapsulateda 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). Tohave concluded that the major shell proteins from date, detailed structural studies have been conducted only onthat 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 cyanobacteriathe Pdu microcompartment, representing the first and some chemoautotrophs by encapsulating essentially all ofstructure 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 (Priceother 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 determinedin the hexamer pore. In view of the hypothesis that to encode proteins necessary for forming a microcompartmentmicrocompartment 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 andof 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 requiredprotein 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 totalspecies 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 theserelatively 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 thatto the proteins that comprise the outer shell of diverse micro- PduA is present in the outer shell (Havemann et al., 2002); thecompartments (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 themicrocompartments 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). The1324 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved
  • 2. StructureStructure 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 (unpublishedFigure 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 evolutionaryChen et al., 1994). The pdu genes are labeled with their respective pdu suffix mechanism for the origin of the observed permutation isdesignations, and drawn in proportion to their lengths and positions in the pdu presented. The resulting novel characteristics of the PduUoperon (Bobik et al., 1999). PduB and PduB0 arise from alternate start sites. hexamer are discussed in light of their potential implicationsDark shaded arrows indicate genes whose encoded proteins are detected inisolated 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 RESULTSBMC shell proteins.(B) A current model of the function of the Pdu microcompartment. The Pdu Structure and Assembly Featuresmicrocompartment 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 andthe 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 Procedurescompartment also contains the enzymatic reactions to generate (biologically and Table S1 available online). The crystal unit cell revealedactive) adenosyl-cobalamin (Ado-B12) from its precursor cob(I)alamin and to two cyclic hexamers in the R3 space group, each sitting on thedisplace inactive deadenosylated B12 derivatives (represented by X-B12) threefold axis of symmetry. There are therefore four independentfrom the diol dehydratase. These reactions are carried out by cob(I)alamin protein chains (A, B, C, and D), two from each hexamer, in theadenosyltransferase (PduO) (Johnson et al., 2001, 2004) and diol dehydratase asymmetric unit. The final atomic model is nearly complete; thereactivase (PduGH), respectively (Bobik et al., 1999). Given this model, theshell 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, anddinucleotide (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 themetabolic 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
  • 3. Structure Structure of a Pdu Shell Protein 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 ofthe 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 adoptsa variation of the typical bacterial microcompartment (BMC) fold (Kerfeld et al., 2005). The 18 amino-terminal and eight carboxy-terminal residuesform novel secondary structural elements. The PduU monomer adopts a compact, wedge-shaped fold ningham et al., 1979). This arrangement is unique among therepresenting 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-dimensionalPduU 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 highermatch those reported for homologous carboxysome shell amino acid sequence identity than alignments with nativeproteins, there are some dramatic differences in folding and PduU. The sequence identity in an alignment to carboxysomeassembly. 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 alignmentsformed extended, two-dimensional molecular layers that are be- (Edgar, 2004) against diverse BMC protein sequences reveallieved to represent the outer shell of the microcompartment (Ker- the conservation of key residues that cannot be aligned usingfeld et al., 2005; Tsai et al., 2007). In contrast, the two indepen- native PduU (Figure 4). For instance, using the permuted PduUdent 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 otherprobably not reflective of the arrangement of BMC protein hex- BMC proteins (e.g., Ile80 and Pro83 in CcmK4); using the nativeamers 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). Theing at their corners. In the hexamer 2 layer, which must be improvement in sequence identity, including key residues widelyspaced equally to the first layer, the edges of neighboring conserved in BMC domain proteins, suggests that PduU ishexamers are parallel, but the spacing is too large to permit inti- related to the typical BMC domain by an effective permutationmate 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, whereviously reported carboxysome hexamers in closely packed residues 109–116 contribute an extra strand to the edge of thearrangements. 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 chainStructural Rearrangements in the BMC Fold terminate on the side of the hexamer opposite from that seenThe 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). Thesecondary structure elements contributed by the C-terminal Central Poreregion of the typical BMC fold are instead contributed by the The N terminus of PduU extends toward the middle of theN-terminal region of PduU. The relationship between the two hexamer, where residues 8–15 from each subunit contribute tocores can therefore be described as a circular permutation (Cun- a parallel six-stranded b-barrel, which is unique among BMC1326 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved
  • 4. StructureStructure of a Pdu Shell Protein 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 interiorbetween 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 stericthe degree of slanting of b strands in a barrel, is 12, which has collisions. Instead, the electron density supports modeling twobeen noted to be a low-energy configuration for six-stranded side chain conformations, which alternate around the barrelb-barrels (Murzin et al., 1994). A similar homo-oligomeric b-bar- (Figures 5A and 5B). In the ‘‘up’’ conformation, the side chainsrel, formed by seven b strands instead of six, is found in the are oriented toward the N-terminal pole of the b-barrel; in theextramembrane 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 conformationalter 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-orientedassembled 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 tightbarrel appears to be too tightly packed with large amino acid packing of methionine and glutamine side chains suggests thatside 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 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved 1327
  • 5. Structure Structure of a Pdu Shell Protein 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 hexamerFigure 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 formGln12 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 moleculespanels 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 representin the configuration observed here. We note that, notwithstand- major constituents of that shell. This property is shared bying the minor differences in amino acid side chain orientations, PduU and one particular carboxysome shell protein, CcmK4the N terminus of PduU adopts nearly equivalent configurations (Heinhorst et al., 2006; Kerfeld et al., 2005). Neither PduU norin the two independent hexamers in the crystal, arguing against CcmK4 is a major constituent of its respective shell (Havemannthe 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 theirWhereas 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. Thesite of the central pore than other structurally characterized need for multiple homologs of the BMC domain proteins—andBMC domain proteins (i.e., those from the carboxysome; Fig- this is the case for every genome in which they have beenure 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 tosion, with Gln12, which is near the C-terminal end of the b-barrel, the speculative suggestion that it could occur at edges of theforming 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 betral 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 theamino acid side chains. In PduU, the loop between residues 76 deep cavity or depression on one side of the hexamer (Figuresand 79 replaces a loop that has three more residues in homolo- 6A and 6B). This open cavity takes the place of a much narrower1328 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved
  • 6. StructureStructure of a Pdu Shell Protein 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 geneFigure 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 andat 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 fromPdu 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 poreGln12, Tyr14, and Phe78 are shown to illustrate their contributions to the surface. that is present in the carboxysome shell protein hexamer (C) is blocked byThe substantial surface contributions by aromatic side chains are a distinction the b-barrel in PduU (B). Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved 1329
  • 7. Structure Structure of a Pdu Shell Protein 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, inRussell, 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 glycolmultiple 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-3350compartment 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 8.2.2ments, Salmonella offers two key features. Protocols have (Berkeley, CA; Table S1).been developed for isolating Pdu microcompartments in intactform (Havemann and Bobik, 2003), and genetic manipulation Structure Determination and Analysisin Salmonella is straightforward. This opens up the possibility Diffraction data were indexed and processed using the programs DENZO andof isolating mutant microcompartments for in vitro experiments; SCALEPACK (Otwinowski and Minor, 1997). The program PHASER (McCoythis has not yet been possible for carboxysomes, which are et al., 2005) was used to solve the structure by molecular replacement usingdifficult 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 COOTproduces 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 mediumcombined biochemical and genetic studies (Stojiljkovic et al., strength setting to the four molecules in the asymmetric unit. Restraints1995). Structure-guided mutagenesis in the Pdu and Eut sys- were released for the last five rounds of the refinement. The final R and Rfreetems 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 usedabout bacterial microcompartments. for visual investigation, molecular measurements, and generation of cartoon structure images of the PduU structure. Measurement bars and labels wereEXPERIMENTAL 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). SuperpositioningTransmission 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 offormed as previously described (Bobik et al., 1999; Havemann and Bobik, structures were calculated over the following chain segments: CcmK22003) using a Zeiss EM-10CA transmission electron microscope (Zeiss; Jena, residues 71–90, CcmK4 residues 73–92, and CsoS1A residues 74–93 overGermany). PduU residues 19–38; CcmK2 residues 4–37, CcmK4 residues 6–39, and CsoS1A residues 8–41 over PduU residues 45–78; and CcmK2 residuesCloning and Protein Purification 41–69, CcmK4 residues 43–71, and CsoS1A residues 45–73 over PduUBriefly, 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 NUMBERSamino 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 accessioncarried 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 fromthe soluble cell fraction by HisTrap HP affinity chromatography (GE Health- SUPPLEMENTAL DATAcare; 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 can0.2 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 ACKNOWLEDGMENTSProtein 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 technicalU.K.) was used to set up crystallization trials by the hanging drop vapor diffu- assistance; Duilio Cascio for crystallographic advice; Morgan Beeby for gel1330 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved
  • 8. StructureStructure of a Pdu Shell Proteinelectrophoresis 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 vivoand Environmental Research of the U.S. Department of Energy, Office of Sci- folding of a circularly permuted jellyroll protein shown by crystal structureence (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 inReceived: March 20, 2008 Salmonella enterica serovar Typhimurium LT2. J. Bacteriol. 185, 5086–5095.Revised: May 29, 2008Accepted: 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.REFERENCES Heinhorst, S., Cannon, G.C., and Shively, J.M. (2006). Carboxysomes and car- boxysome-like inclusions. In Complex Intracellular Structures in Prokaryotes,Adkins, J.N., Mottaz, H.M., Norbeck, A.D., Gustin, J.K., Rue, J., Clauss, T.R., J.M. Shively, ed. (Berlin: Springer-Verlag), pp. 141–165.Purvine, S.O., Rodland, K.D., Heffron, F., and Smith, R.D. (2006). Analysisof the Salmonella typhimurium proteome through environmental response Heithoff, D.M., Conner, C.P., Hentschel, U., Govantes, F., Hanna, P.C., andtoward infectious conditions. Mol. Cell. Proteomics 5, 1450–1461. Mahan, M.J. (1999). Coordinate intracellular expression of Salmonella genes induced during infection. J. Bacteriol. 181, 799–807.Bass, R.B., Strop, P., Barclay, M., and Rees, D.C. (2002). Crystal structure ofEscherichia coli MscS, a voltage-modulated and mechanosensitive channel. Iancu, C.V., Ding, H.J., Morris, D.M., Dias, D.P., Gonzales, A.D., Martino, A.,Science 298, 1582–1587. and Jensen, G.J. (2007). The structure of isolated synechococcus strain WH8102 carboxysomes as revealed by electron cryotomography. J. Mol.Bobik, T.A. (2006). Polyhedral organelles compartmenting bacterial metabolic Biol. 372, 764–773.processes. Appl. Microbiol. Biotechnol. 70, 517–525. Jensen, T.E., and Bowen, C.C. (1961). Organization of the centroplasm inBobik, T.A., Ailion, M., and Roth, J.R. (1992). A single regulatory gene inte- Nostoc pruniforme. Iowa Acad. Sci. Proc. 68, 86–89.grates control of vitamin B12 synthesis and propanediol degradation. J. Bac-teriol. 174, 2253–2266. Johnson, C.L., Pechonick, E., Park, S.D., Havemann, G.D., Leal, N.A., and Bo- bik, T.A. (2001). Functional genomic, biochemical, and genetic characteriza-Bobik, T.A., Xu, Y., Jeter, R.M., Otto, K.E., and Roth, J.R. (1997). Propanediol tion of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferaseutilization genes (pdu) of Salmonella typhimurium: three genes for the propane- gene. J. Bacteriol. 183, 1577–1584.diol dehydratase. J. Bacteriol. 179, 6633–6639. Johnson, C.L., Buszko, M.L., and Bobik, T.A. (2004). Purification and initialBobik, T.A., Havemann, G.D., Busch, R.J., Williams, D.S., and Aldrich, H.C. characterization of the Salmonella enterica PduO ATP:Cob(I)alamin adenosyl-(1999). The propanediol utilization (pdu) operon of Salmonella enterica serovar transferase. J. Bacteriol. 186, 7881–7887.Typhimurium LT2 includes genes necessary for formation of polyhedral organ-elles involved in coenzyme B(12)-dependent 1, 2-propanediol degradation. J. Kerfeld, C.A., Sawaya, M.R., Tanaka, S., Nguyen, C.V., Phillips, M., Beeby, M.,Bacteriol. 181, 5967–5975. and Yeates, T.O. (2005). Protein structures forming the shell of primitive bacterial organelles. Science 309, 936–938.Brinsmade, S.R., Paldon, T., and Escalante-Semerena, J.C. (2005). Minimal Klumpp, J., and Fuchs, T.M. (2007). Identification of novel genes in genomicfunctions and physiological conditions required for growth of Salmonella islands that contribute to Salmonella typhimurium replication in macrophages.enterica on ethanolamine in the absence of the metabolosome. J. Bacteriol. Microbiology 153, 1207–1220.187, 8039–8046. Koprowski, P., and Kubalski, A. (2003). C termini of the Escherichia coli me-Chen, P., Andersson, D.I., and Roth, J.R. (1994). The control region of the pdu/ chanosensitive ion channel (MscS) move apart upon the channel opening. J.cob regulon in Salmonella typhimurium. J. Bacteriol. 176, 5474–5482. Biol. Chem. 278, 11237–11245.Collaborative Computational Project, Number 4 (1994). The CCP4 suite: Krissinel, E., and Henrick, K. (2004). Secondary-structure matching (SSM),programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. a new tool for fast protein structure alignment in three dimensions. Acta50, 760–763. Crystallogr. D Biol. Crystallogr. 60, 2256–2268.Colovos, C., and Yeates, T.O. (1993). Verification of protein structures: pat- Leal, N.A., Havemann, G.D., and Bobik, T.A. (2003). PduP is a coenzyme-a-terns of nonbonded atomic interactions. Protein Sci. 2, 1511–1519. acylating propionaldehyde dehydrogenase associated with the polyhedralConner, C.P., Heithoff, D.M., Julio, S.M., Sinsheimer, R.L., and Mahan, M.J. bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella(1998). Differential patterns of acquired virulence genes distinguish Salmonella enterica serovar Typhimurium LT2. Arch. Microbiol. 180, 353–361.strains. Proc. Natl. Acad. Sci. U.S.A. 95, 4641–4645. Lee, B., and Richards, F.M. (1971). The interpretation of protein structures:Cunningham, B.A., Hemperly, J.J., Hopp, T.P., and Edelman, G.M. (1979). Fa- estimation of static accessibility. J. Mol. Biol. 55, 379–400.vin versus concanavalin A: circularly permuted amino acid sequences. Proc. McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C., and Read, R.J. (2005).Natl. Acad. Sci. U.S.A. 76, 3218–3222. Likelihood-enhanced fast translation functions. Acta Crystallogr. D Biol.Drews, G., and Niklowitz, W. (1956). Cytology of cyanophycea. II. Centroplasm Crystallogr. 61, 458–464.and granular inclusions of Phormidium uncinatum. Arch. Mikrobiol. 24, Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macro-147–162. molecular structures by the maximum-likelihood method. Acta Crystallogr.Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy D Biol. Crystallogr. 53, 240–255.and high throughput. Nucleic Acids Res. 32, 1792–1797. Murzin, A.G., Lesk, A.M., and Chothia, C. (1994). Principles determining theEmsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular structure of beta-sheet barrels in proteins. I. A theoretical analysis. J. Mol.graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132. Biol. 236, 1369–1381.Ferguson, K.A. (1964). Starch-gel electrophoresis—application to the classifi- Otwinowski, Z., and Minor, W. (1997). Processing of X-ray diffraction datacation of pituitary proteins and polypeptides. Metabolism 13 (Suppl ), collected in oscillation mode. Methods Enzymol. 276, 307–326.985–1002. Ponting, C.P., and Russell, R.B. (1995). Swaposins: circular permutationsGantt, E., and Conti, S.F. (1969). Ultrastructure of blue-green algae. J. Bacter- within genes encoding saposin homologues. Trends Biochem. Sci. 20, 179–iol. 97, 1486–1493. 180. Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved 1331
  • 9. Structure Structure of a Pdu Shell ProteinPrice, G.D., Coleman, J.R., and Badger, M.R. (1992). Association of carbonic tide CsoS1 of the thiobacilli are present in cyanobacteria and enteric bacteriaanhydrase activity with carboxysomes isolated from the cyanobacterium that form carboxysomes-polyhedral bodies. Can. J. Bot. 76, 906–916.Synechococcus PCC7942. Plant Physiol. 100, 784–793. Stojiljkovic, I., Baumler, A.J., and Heffron, F. (1995). Ethanolamine utilizationSampson, E.M., and Bobik, T.A. (2008). Microcompartments for B12-depen- in Salmonella typhimurium: nucleotide sequence, protein expression, anddent 1,2-propanediol degradation provide protection from DNA and cellular mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster.damage by a reactive metabolic intermediate. J. Bacteriol. 190, 2966–2971. J. Bacteriol. 177, 1357–1366.Schmid, M.F., Paredes, A.M., Khant, H.A., Soyer, F., Aldrich, H.C., Chiu, W.,and Shively, J.M. (2006). Structure of Halothiobacillus neapolitanus carboxy- Tanaka, S., Kerfeld, C.A., Sawaya, M.R., Cai, F., Heinhorst, S., Cannon, G.C.,somes by cryo-electron tomography. J. Mol. Biol. 364, 526–535. and Yeates, T.O. (2008). Atomic-level models of the bacterial carboxysomeSchulz, G.E. (2002). The structure of bacterial outer membrane proteins. shell. Science 319, 1083–1086.Biochim. Biophys. Acta 1565, 308–317. Tsai, Y., Sawaya, M.R., Cannon, G.C., Cai, F., Williams, E.B., Heinhorst, S.,Shively, J.M., Decker, G.L., and Greenawalt, J.W. (1970). Comparative ultra- Kerfeld, C.A., and Yeates, T.O. (2007). Structural analysis of CsoS1A and thestructure of the Thiobacilli. J. Bacteriol. 101, 618–627. protein shell of the Halothiobacillus neapolitanus carboxysome. PLoS Biol.Shively, J.M., Ball, F., Brown, D.H., and Saunders, R.E. (1973). Functional or- 5, e144.ganelles in prokaryotes: polyhedral inclusions (carboxysomes) of Thiobacillusneapolitanus. Science 182, 584–586. Yeates, T.O., Tsai, Y., Tanaka, S., Sawaya, M.R., and Kerfeld, C.A. (2007). Self-Shively, J.M., Bradburne, C.E., Aldrich, H.C., Bobik, T.A., Mehlman, J.L., Jin, assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells.S., and Baker, S.H. (1998). Sequence homologs of the carboxysomal polypep- Biochem. Soc. Trans. 35, 508–511.1332 Structure 16, 1324–1332, September 10, 2008 ª2008 Elsevier Ltd All rights reserved