We are studying the relationship between protein structure and function using three different types of proteins: antifreeze proteins (AFPs), which help organisms to resist or tolerate freezing; bacterial adhesins, which play a role in biofilm formation and infection; and calpains, enzymes that selectively cut proteins within the cell in response to calcium signals. (Funded by CIHR, NSERC and MDA.)

Antifreeze proteins are found in some fishes, insects, plants and microorganisms. They bind to ice crystals and prevent them from growing to a size where they would damage the host. Our research involves the isolation and characterization of antifreeze proteins from different sources, the study of their evolution, and the cloning and expression of their genes to produce recombinant proteins for 3-D structural analysis by NMR and/or X-ray crystallography. AFPs are proving to have remarkably diverse structures. We are trying to identify their ice-binding sites/residues using site-directed mutagenesis in order to learn more about their mechanism(s) of action and what structural features are required for binding to ice. We are also engineering superior AFPs based on this information, and are applying them to the sub-zero storage of organs, tissues and cells. Another of our research interests is in ice-nucleation proteins (INPs) and how they relate to antifreeze proteins. More

Bacterial adhesins are long, thin proteins attached to the outer membrane of bacteria, which anchor their hosts to various surfaces where the bacteria can form biofilms. Structural characterization and functional analysis of all the adhesins are revealing methods for blocking biofilm formation that will be useful in preventing infections. More

Calpains are complex, multi-domain calcium-dependent proteases involved in calcium signaling. We are studying their mechanism of activation and inhibition, and are using peptide and combinatorial compound libraries to develop calpain-specific inhibitors and substrates. The latter will be used to better define the physiological roles of calpain, and as leads for developing drugs to help prevent the calpain-mediated damage associated with heart attacks, stroke, neurodegeneration, and muscular dystrophy. More


Frost damage: In the photograph below, cold air flowing off a roof has caused frost damage to hydrangea bushes, causing them to turn brown. But leaves alongside, which were under the eaves of the roof, remain green because they were sheltered from the cold air and escaped frost damage. The hydrangea leaves are populated by bacteria like Pseudomonas syringae and Pseudomonas borealis that have ice nucleation proteins (INPs) in their outer membrane. These INPs will nucleate freezing at just a couple of degrees below zero. In this example, a small temperature difference in the microenvironment has made the difference between freezing and not freezing. It is thought that these bacteria may have developed INPs to help gain access to nutrients from the plants when they are damaged by frost.

How do INPs nucleate freezing? Unfortunately, we do not yet have a structure of an INP to help figure out how they work. However, we and others have modeled the central repeating section (yellow region in Figure 1 below) of these >100-kDa proteins as a beta-solenoid with 16-residue coils. This region has a similar tertiary structure to many antifreeze proteins. Also, many of the INP coils have Thr-X-Thr motifs, which are the same as those found on the ice-binding sites some of the most active antifreeze proteins.

Figure 1








If we are right in thinking that the ice-binding sites use these Thr-X-Thr motifs to organize water into an ice-like pattern that binds the protein to ice, then the function of these motifs in INPs might be to organize so much ice-like water that it starts the nucleation process.

In addition to the INPs being much larger than antifreeze proteins, the INPs seem to aggregate on the surface. This is nicely illustrated by a construct made in Dr. Virginia Walker’s lab in the Biology Department at Queen’s University, where the INP has been tagged with green fluorescent protein. This shows that the INPs accumulate at the poles of the E. coli bacteria, which are ~2 microns long (Figure 2).   

Figure 2


We are trying to test the hypothesis that INPs are large version of antifreeze proteins and that they work by a similar mechanism.


Structure: Bacterial adhesins are long filamentous proteins that attach host bacteria to surfaces. Attachment is an early event in formation of biofilms. Guo et al., 2017 have recently pieced together the first complete structure of an RTX adhesin. This giant 1.5 MDa protein is a single polypeptide chain that folds into ~130 domains with different functions in different regions along the chain (Fig. 1). At the C terminus is the Type I secretion signal that directs the export of the adhesin. Next, is the ligand-binding region, which in this example has domains to bind ice, terminal sugars, and a specific peptide sequence. The majority of the adhesin domains are in the extender (or stalk) region that puts distance between the ligand-binding domains and the N-terminal membrane-anchoring region, which is a novel structure responsible for retention of the adhesin in the outer membrane of its bacterial host. Bioinformatic analyses of other RTX adhesins has shown that they adopt similar structures, but with different binding domains and different lengths.

Fig. 1 Domain structure of the ice-binding RTX adhesin from a marine bacterium








Function: This particular adhesin comes from a marine bacterium (Marinomonas primoryensis) isolated from a salt water lake in Antarctica that is covered in ice for most of the year. We reasoned that this aerobic bacterium has developed an ice-binding domain at the end of the adhesin to locate itself just under the ice where photosynthetic microorganisms like algae and diatoms get the most sunlight (Fig. 2 left panel). But what are the functions of the other ligand-binding domains? It turns out that they bind to the surface of an Antarctic diatom, Chaetoceros neogracile – one of the diatoms we received from our colleague, Dr. Eon-Seon Jin in S. Korea. The motile M. primoryensis home in on the diatoms and collectively swim them to the ice where they attach the cell mass via the ice-binding domain on the adhesin to form a mixed microorganism biofilm (Fig. 2 right panel). This is a neat example of symbiosis. The non-motile diatoms are brought by the bacteria to the best place for photosynthesis. The bacteria benefit by receiving oxygen and other metabolic waste products from the diatoms.


Fig. 2 Marinomonas primoryensis binding to ice and diatoms to form a mixed species biofilm under marine ice in Antarctica


Applications: We are being guided by this model system to deduce how other RTX adhesins bind their host bacteria to different surfaces. Some of these bacteria are human and animal pathogens. By learning how to block these interactions we might be able to better control bacterial infections and biofilm formation.  


Calpains are complex, multi-domain proteases involved in calcium signaling in the cell. We are interested in the mechanism by which these enzymes sense calcium and become activated to make specific cuts in target proteins. In addition to the ubiquitous m- and mu-calpain isoforms, a dozen tissue-specific variants have been recognized, all of which share a similar papain-like protease core.


Conformational change in the protease core of calpain during activation by calcium. (3.3 Mb)





The active site cleft is realigned by the cooperative binding of two calcium ions (gold spheres) (Moldoveanu et al., 2002). The realignment is shown by the morphing of ten images and appears as a rotation of domain II (left) relative to domain I (right).

Courtesy of Dr. Rob Campbell

CALPAINS - calcium-dependent cysteine proteases

Fig. 1. Model of calpain showing the known calcium binding sites




This diagram is a composite of the two ubiquitous mammalian calpain isoforms, mu-calpain and m-calpain. The protease core (bounded by the dotted line) is the papain-like region of calpain. It contains two domains (I&II) in blue and cyan, respectively. In the calcium-free form (not shown), the active site cleft is wedged open into a catalytically inactive configuration. We have recently discovered two novel calcium-binding sites, one on either side of the cleft (Moldoveanu et al. 2002). When calcium (yellow or red spheres) cooperatively binds to these sites, the wedge is removed, and the cleft closes into its active form shown here.

The protease core is common to all calpains and is their defining characteristic. In mu- and m-calpain it is followed in the large (80 kDa) by two other domains: domains III (green) and IV (yellow). Domain III is also thought to bind calcium and is structurally similar to a membrane-binding domain found in some phopholipases and protein kinases. The C-terminal domain (IV) contains five EF-hand motifs (yellow) and binds three or four calcium ions. The fifth EF-hand is used as a dimerization interface to bind to the equivalent region of domain VI. This second penta-EF-hand domain (copper) is the C-terminal portion of the small subunit. The N-terminal region (not shown) is an unstructured glycine-rich sequence. The circular arrangement of domains is completed by a contact between the N-terminal anchor helix (red cylinder) and domain VI. 

Moldoveanu, T., Hosfield, C.M., Lim, D., Elce, J.S., Jia, Z. and Davies, P.L. “A Ca2+ Switch Aligns the Active Site of Calpain”. (2002) Cell 108, 649-660.

Moldoveanu, T., Hosfield, C.M., Jia, Z., Elce, S.J. and Davies, P.L. “Ca2+-induced structural changes in rat m-calpain revealed by partial proteolysis”. (2001) Biochim. Biophys. Acta 1545, 245-254.

We are grateful to the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council (NSERC) and the Muscular Dystrophy Association (MDA) for funding our research.


Queen's University

Kingston Ontario Canada

© 2018  Peter L. Davies

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