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Fig. 2. Structure of the InlB LRR region (residues 77–242). The right-handed coil of the LRR alternates between β-strands and 310-helices. The β-strands form the concave face of the molecule and have a superhelical twist not observed in other LRR proteins. This figure and Fig. 4 were generated with MOLSCRIPT (48) and rendered with RASTER3D (49).

convex face of the molecule (Fig. 2). This curvature would appear to limit the possible number of tandem LRRs, because a closed circle would be formed. However, the InlB β-strands are twisted and give the entire structure a right-handed superhelical twist, thereby placing no obvious limits on the possible number of tandem repeats (39). The β-strands of RI, U2LRR, and rnalp are not as extensively twisted and indeed place a limit on the number of possible tandem repeats. It is not clear at present what gives rise to this difference.

The coil regions connecting β-strands and 310-helices constitute the great majority (14–17 residues) of the 22-residue InlB LRR. Even though these regions lack conventional secondary structure, they form highly regular structures (rms deviation of 0.32 Å in Cα positions). This regularity may in part be caused by the presence of water molecules that act as extensions of secondary structure. These waters are intercalated between repeats and form bridging hydrogen bonds between main-chain atoms of adjacent repeats. Structural determination of InlB to high resolution (1.86 Å) allowed visualization of three distinct spines of water molecules that run the length of the repeat region (39). Some of these hydrogen bond-mediating waters are also observed in the 2.0-Å resolution structure of human RI (42). A regular pattern of hydrogen bond-mediating waters is expected to be present in other LRR proteins and to be observed in high-resolution structures of other LRR proteins.

LRR Flanking Sequences

The LRR regions are flanked by sequences that are conserved in the internalin family and that most likely play a role in providing stability. At the N terminus of the LRR region is a segment of ≈40 residues termed the “N-terminal cap” in InlB, because it provides a hydrophilic cap on the hydrophobic core of the first LRR (39). The hydrophobic core would be exposed to solvent at either end of the LRR were it not for other portions of the protein. The N-terminal cap forms numerous polar and hydrophobic interactions with the first LRR. The most prominent of these is a hydrophobic interaction with Tyr at position 20 of the first LRR, a highly conserved residue in the internalin family (Fig. 1).

In addition to its structural role, the N-terminal cap has been suggested to be involved in function based on the observation of two highly solvent-exposed calcium ions that bind to this region (39). The calciums are not required for formation of protein structure and may instead act as metal ion bridges between InlB and mammalian cell surface receptors or binding proteins. The N-terminal cap and LRR regions of InlB are sufficient to elicit mammalian cell effects and to induce phagocytosis (38).

At the C terminus of the LRR region is a conserved sequence of ≈100 residues termed the interrepeat (IR) region (17). The IR region is dispensable for the mammalian cell signaling activities of InlB but is required in InlA for inducing phagoctyosis (37). The IR region has not yet been visualized, but crystals of intact InlB have been obtained that should elucidate the role of this as well as other regions of internalins (M.M. and P.G., unpublished data). At least a portion of the IR region is likely to provide a hydrophilic cap on the last LRR.

LRR Pattern: Structural Residues

A total of 10 positions of the 22-residue internalin LRR are highly conserved and serve structural rather than functional roles. Seven positions (2, 5, 7, 12, 15, 18, and 21) of the repeat contain mainly Leus or Iles that point inward and compose the hydrophobic core of the protein (Fig. 3). Other hydrophobic residues are accommodated at these positions, including Val, Phe, Met, and Ala (Fig. 1). Position 10 also faces inward and usually contains Asns or Glns, which form the so-called “Asn ladder” (45). The Asn hydrogen bonds to main-chain atoms in its own repeat and to those in the preceding repeat. This explains why position 10 in the first repeat is not constrained to be an Asn or Gln.

The seven hydrophobic positions along with the Asn-ladder position define the internalin LRR sequence motif (Fig. 4). This motif corresponds almost identically to the 22-residue repeat

Fig. 3. Structure of a single InlB LRR. Positions 2, 5, 7, 10, 12, 14, 15, 17, 18, and 21 are conserved for structural reasons. The remaining positions are solvent exposed and variable. The register of the InlB LRR is that described for porcine RI (40).



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