The Beta Helix of P. aeruginosa Alkaline Protease

Pseudomonas aeruginosa secretes two different metal-containing proteases. One of these is a 50 kDa alkaline protease (AP) belonging to the serralysin family. This protease is zinc- dependent and requires several calcium ions for stabilization of the folded structure.

The first 17 residues of the AP form an alpha helix

Residues 18-250 form a catalytic domain with a single Zn ion at the active site.

There is an alpha helix near the active site Zn .

The C-terminal end of this helix is part of a Zn-binding motif, H(176)EXXHXXGXXH(186) that surrounds the zinc ion. The three His residues in this motif are Zn ligands.

The Zn coordination sphere is a trigonal bipyramid, with His-176 (yellow), His-186(orange), and a water molecule (red) lying in the trigonal plane.

The apices of the trigonal bipyramid are somewhat distorted and consist of His-180 (top) and Tyr-216 (bottom).

The more interesting part of the alkaline protease in many ways is the C-terminal domain.

One of these beta sheets lies along one side of the C-terminal domain.

A second beta sheet faces the first one, forming a beta sandwich, with the two faces of the sandwich connected by loop structures.

Interestingly, this domain of the AP binds eight different calcium ions.

Five of these Ca ions are enclosed within the central region of the beta sandwich.

This 'beta sandwich' encloses an even more interesting structure - a 'beta coil', which has also been dubbed a 'beta helix'.

Take another look at the beta helix as it rotates.

Now take a look at the 'beta helix' from a different perspective.

The so-called helix is approximately elliptical in cross-section, with Ca ions nestled in the loop.

This interesting structure is formed from six repeats of a consensus Ca-binding sequence.

Each of these consensus sequences consists of a three-residue beta strand, followed by a loop that surrounds the Ca ion either above it or below it.

The 3-residue beta strand is usually hydrophobic and the middle residue of the three is a consensus leucine.

The Ca loop consists of three glycine residues in a GGXG motif.

The last residue in the 9-residue motif is almost always Asp (sometimes Asn), whose side chain carboxyl (or amide) coordinates the Ca ion.

Here is a view of how a single loop coordinates one of the Ca ions.

The complete, 9-residue consensus sequence is thus ULUGGXGXD, where U is usually hydrophobic and X can be any residue.

Let's now consider the coordination of Ca ions in this structure in more detail.

Ca ions in the beta helix are fully coordinated by two adjacent loops.

The Ca coordination geometry is approximately octahedral, with six protein ligands arranged around the metal ion.

Most Ca ions bound to proteins possess one or more water ligands in their first coordination sphere, but Ca ions in the beta helix motif are 'dry'! None of the Ca ions in the helix possesses even a single water. All metal ligands are provided by the protein.

Let's take a last look at the beta helix motif.

Where they have been found in proteins, beta helix motifs are decidedly hydrophobic. Notice the hydrophobic residues (yellow) surrounding this structure.

Another interesting aspect of the beta helix is the manner in which the consensus Leu residues fill the inside of the structure - between pairs of Ca ions.

Here is a view of the hydrophobic surface of the beta helix motif.

The beta helix motif typically contains only a few polar residues. In the present 45-residue structure, there are only 11 polar residues, and 4 of these are the inward-facing Asp(Asn) residues that coordinate Ca. This leaves only 7 outward-facing polar residues.

The beta helix is an unusual structure. Though filled with polar Ca ions, the overall structure is highly hydrophobic. As such, it may represent a means for inserting a protein into and across a biological membrane. In this regard, it is interesting to note that the adenylyl cyclase toxin (ACT) produced by Bordetella pertussis contains 38 repeats of the ULUGGXGXD motif with approximately the same number of tightly-bound, waterless Ca ions! ACT functions by inserting itself across host-cell membranes, so that the adenylyl cyclase domain can alter cellular metabolism by producing large amounts of cAMP. A large beta helix filled with Ca ions would make a suitable device for such a membrane-crossing event.