Structure of Spy0129.

<p>A) Stereo view of the monomer (molecule B), colour coded from N-terminus (blue) to C-terminus (red). The putative catalytic residues Cys221 and His126 are shown in stick representation. B) Topology diagram for Spy0129, coloured as in A) and labelled from α1 to α6 for α-helices and β1 to β8 for β-strands.</p

Sequence alignment of Spy0129 with other class B sortases.

<p>Invariant residues are highlighted in dark blue and conserved residues in lighter blue colours. Putative catalytic residues are coloured in purple. The secondary structure elements from Spy0129 are shown above the sequence. Residues corresponding to the sortase signature motif are boxed with a black outline. The β6/β7 loop region of <i>S. aureus</i> SrtB which was shown to confer substrate specificity is outlined with a dotted red box.</p

Comparison of different sortase structures.

<p>A) Class B: Spy0129 from <i>S. pyogenes</i> (this work); B) Class B: SrtB from <i>S. aureus</i> (PDB code 1T2P): C) Class C: SrtC1 from <i>S. pneumoniae</i> (PDB code 2W1J); and D) Class A: SrtA-LPAT* complex structure from <i>S.aureus</i> (PDB code 2KID). In each case, the β6/β7 region is highlighted with darker colour than the rest of the molecule. The lockable lid in the pneumococcal SrtC1 is highlighted in blue. All four structures are shown in equivalent orientations. The catalytic Cys residues are shown in stick mode.</p

Active site of Spy0129.

<p>Stereo view of the active site region, with molecule A (magenta) superimposed on to molecule B (light teal) to show the conformational differences in the β4/β5 and β7/β8 loops. The residues Cys221, His126, His127 and Arg229 are shown in stick mode, coloured to correspond to the molecule to which they belong. In molecule A, Cys221 is linked to His127 through a bound zinc ion (magenta sphere) and His126 is oriented away from Cys221. In molecule B, Cys221 is linked to His126 through a zinc atom (blue sphere) and His127 is oriented away.</p

Surface structures involved in substrate binding.

<p>Stereo diagram of Spy0129 (magenta), with its β6/β7 region highlighted in darker magenta. Superimposed on to Spy0129 are the lockable lid present in the class C SrtC enzymes from <i>S. pneumoniae</i> (dark blue) and β6/β7 region from <i>S. aureus</i> SrtA (dark gray) including the 3₁₀-helix that helps bind the LPAT* peptide analogue.</p

Data collection, structure determination and refinement statistics.

a<p>Values in parentheses are for outermost shell.</p

Structure and Activity of <i>Streptococcus pyogenes</i> SipA: A Signal Peptidase-Like Protein Essential for Pilus Polymerisation

<div><p>The pili expressed on the surface of the human pathogen <i>Streptococcus pyogenes</i> play an important role in host cell attachment, colonisation and pathogenesis. These pili are built from two or three components, an adhesin subunit at the tip, a major pilin that forms a polymeric shaft, and a basal pilin that is attached to the cell wall. Assembly is carried out by specific sortase (cysteine transpeptidase) enzyme. These components are encoded in a small gene cluster within the <i>S. pyogenes</i> genome, often together with another protein, SipA, whose function is unknown. We show through functional assays, carried out by expressing the <i>S. pyogenes</i> pilus components in <i>Lactococcus lactis</i>, SipA from the clinically important <i>M1T1</i> strain is essential for pilus assembly, and that SipA function is likely to be conserved in all <i>S. pyogenes</i>. From the crystal structure of SipA we confirm that SipA belongs to the family of bacterial signal peptidases (SPases), which process the signal-peptides of secreted proteins. In contrast to a previous arm-swapped SipA dimer, this present structure shows that its principal domain closely resembles the catalytic domain of SPases and has a very similar peptide-binding cleft, but it lacks the catalytic Ser and Lys residues characteristic of SPases. In SipA these are replaced by Asp and Gly residues, which play no part in activity. We propose that SipA functions by binding a key component at the bacterial cell surface, in a conformation that facilitates pilus assembly.</p></div

Western immunoblots of <i>L. lactis</i> expressing <i>S. pyogenes</i> pili.

<p>Cell wall extracts from <i>L. lactis</i> cells transformed with plasmids carrying the <i>S. pyogenes</i> M1/T1 strain pilus operon were immunoblotted with antisera against either FctA backbone pilin (anti-Spy0128), FctB minor pilin (anti-Spy0130), or Cpa adhesin (anti-Spy0125) to show the effect of SipA mutations on pilus polymerisation. Cells expressing WT levels of SipA show a laddering of high molecular weight polymers. SipA deletion mutant (ΔSipA) shows no pilin polymerisation, only monomeric FctA or FctB. Mutating aspartic acid D61 (SipA D61A and D61S) or the double mutation of SipA D61A/K98A, or the V99R S3 pocket-occluding mutant have minimal effect on pilus polymerisation, with each mutant producing high molecular weight polymers. Replacement of the M1/T1 SipA (FCT2) with T9 SipA (FCT3) produced pilus polymerisation, but with more lower molecular weight multimers including FctA-B dimers (∼50 kDa) and pili with no cpa. FctA = 32 kDa, FctB = 18 kDa, Cpa = 75 kDa.</p

Comparison of SipA and <i>E. coli</i> SPase-I.

<p>(<b>A</b>) Stereo-view of a structural alignment between the extracellular domains of SipA and SPase-I. The conserved catalytic core domain of SipA and SPase-I is shown in green and magenta, respectively, and the non-catalytic 'cap' domains in blue (SipA) and yellow (SPase-I). Shown in stick form are the SPase-I catalytic dyad residues (Ser 90 and Lys 145) and the corresponding residues in SipA (Asp 48 and Gly 85, and the nearby Lys 83). An arrow depicts the position of the peptide binding clefts. (<b>B</b>) Topology diagrams of SipA and <i>E. coli</i> SPase-I, color-coded as for the ribbon diagram. Dashed lines represent regions not visible in the electron density. The positions of key catalytic residues are shown in circles. N = N-terminus, C = C-terminus.</p

Comparison of the SipA and SPase-I substrate binding pockets.

<p>Surface representation of the substrate binding pockets of (<b>A</b>) <i>E. coli</i> SPase-I (PDB ID, 3IIQ) and (<b>B</b>) SipA. The molecular surface is colored red for residues involved in the catalytic center of SPase-I and the corresponding residues in SipA; orange for residues contributing side chain atoms to the S1 and S2 pocket; yellow for those residues contributing side chain atoms to the S3 pocket; and purple for residues bridging the two pockets. The SipA A' peptide (cyan) and arylomycin (yellow) are shown in stick form bound to SipA and SPase-I, respectively. (<b>C</b>) Superposition of the active sites of SipA and SPase-I showing hydrogen bond interactions. SipA residues are listed in black with large dashes, and SPase-I residues are in red with small dashes. Homologous residues are grouped. A' peptide (Gly -2 to Phe 39, cyan) and arylomycin (fatty acid tail not included, yellow) are shown in stick form as a side view in the substrate binding pocket, colored by element (carbon, cyan or yellow; oxygen, red; nitrogen, blue). A surface representation of the SipA pocket showing the S1 and S3 pockets is in green.</p