Ice-binding proteins (IBPs) are found in many biological kingdoms where they protect organisms from freezing damage as antifreeze agents or inhibitors of ice recrystallization. Here, the crystal structure of recombinant IBP from carrot (Daucus carota) has been solved to a resolution of 2.3 Å. As predicted, the protein is a structural homologue of a plant polygalacturonase-inhibiting protein forming a curved solenoid structure with a leucine-rich repeat motif. Unexpectedly, close examination of its surface did not reveal any large regions of flat, regularly spaced hydrophobic residues that characterize the ice-binding sites (IBSs) of potent antifreeze proteins from freeze-resistant fish and insects. An IBS was defined by site-directed mutagenesis of residues on the convex surface of the carrot solenoid. This imperfect site is reminiscent of the irregular IBS of grass 'antifreeze' protein. Like the grass protein, the carrot IBP has weak freezing point depression activity but is extremely active at nanomolar concentrations in inhibiting ice recrystallization. Ice crystals formed in the presence of both plant proteins grow slowly and evenly in all directions. We suggest that this slow, controlled ice growth is desirable for freeze tolerance. The fact that two plant IBPs have evolved very different protein structures to affect ice in a similar manner suggests this pattern of weak freezing point depression and strong ice recrystallization inhibition helps their host to tolerate freezing rather than to resist it.

Antifreeze proteins (AFPs) are characterized by their ability to adsorb to the surface of ice crystals and prevent any further crystal growth. AFPs have independently evolved for this purpose in a variety of organisms that encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures, it has been suggested that all AFPs can organize ice-like water patterns on one side of the protein (the ice-binding site) that helps bind the AFP to ice. Here, to test this hypothesis, we have solved the crystal structure at 2.05 Å resolution of an AFP from the longhorn beetle, Rhagium mordax with five molecules in the unit cell. This AFP is hyperactive, and its crystal structure resembles that of the R. inquisitor ortholog in having a β-solenoid fold with a wide, flat ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the R. inquisitor AFP crystallized with its ice-binding site (IBS) making protein–protein contacts that limited the surface water patterns. Whereas the R. mordax AFP crystallized with the IBSs exposed to solvent enabling two layers of unrestricted ordered surface waters to be seen. These crystal waters make close matches to ice lattice waters on the basal and primary prism planes.

With better tools for data processing and with synchrotron beamlines that are capable of collecting data at longer wavelengths, sulfur-based native single-wavelength anomalous dispersion (SAD) phasing has become the `first-choice' method for de novo protein structure determination. However, for many proteins native SAD phasing can be simplified by taking advantage of their interactions with natural metal cofactors that are stronger anomalous scatterers than sulfur. This is demonstrated here for four unique domains of a 1.5 MDa calcium-dependent adhesion protein using the anomalous diffraction of the chelated calcium ions. In all cases, low anomalous multiplicity X-ray data were collected on a home-source diffractometer equipped with a chromium rotating anode (λ = 2.2909 Å). In all but one case, calcium SAD phasing alone was sufficient to allow automated model building and refinement of the protein model after the calcium substructure had been determined. Given that Ca atoms will be present in a significant percentage of proteins that remain uncharacterized, many aspects of the data-collection and processing methods described here could be broadly applied for routine de novo structure elucidation.

Calpains are intracellular, calcium-activated cysteine proteases. Calpain-3 is abundant in skeletal muscle, where its mutation-induced loss of function causes limb-girdle muscular dystrophy type 2A. Unlike the small subunit-containing calpain-1 and -2, the calpain-3 isoform homodimerizes through pairing of its C-terminal penta-EF-hand domain. It also has two unique insertion sequences (ISs) not found in the other calpains: IS1 within calpain-3's protease core and IS2 just prior to the penta-EF-hand domain. Production of either native or recombinant full-length calpain-3 to characterize the function of these ISs is challenging. Therefore, here we used recombinant rat calpain-2 as a stable surrogate and inserted IS1 into its equivalent position in the protease core. As it does in calpain-3, IS1 occupied the catalytic cleft and restricted the enzyme's access to substrate and inhibitors. Following activation by Ca2+, IS1 was rapidly cleaved by intramolecular autolysis, permitting the enzyme to freely accept substrate and inhibitors. The surrogate remained functional until extensive intermolecular autoproteolysis inactivated the enzyme, as is typical of calpain-2. Although the small-molecule inhibitors E-64 and leupeptin limited intermolecular autolysis of the surrogate, they did not block the initial intramolecular cleavage of IS1, establishing its role as a propeptide. Surprisingly, the large-molecule calpain inhibitor, calpastatin, completely blocked enzyme activity, even with IS1 intact. We suggest that calpastatin is large enough to oust IS1 from the catalytic cleft and take its place. We propose an explanation for why calpastatin can inhibit calpain-2 bearing the IS1 insertion but cannot inhibit WT calpain-3.

Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca2+-activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys129 calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca2+ ions to the equivalent sites used in calpain-1 and -2. These structures of the calpain-3 active site with residual IS1 and with bound E-64 and leupeptin may help guide the design of calpain-3-specific inhibitors.

Antifreeze proteins (AFPs) are characterized by their capacity to adsorb to the surface of ice crystals and prevent their growth. This adsorption lowers the freezing temperature of a solution below its melting point. AFPs have independently evolved in a variety of organisms that may encounter the threat of freezing, including many species of polar fish, insects, plants and microorganisms. Despite their diverse origins and structures we suggest that all AFPs organize ice-like water patterns on one side of the protein (the icebinding site) that then bind the AFP to ice. Here, to help test this hypothesis, we have solved two AFP crystal structures. One is of Lake Ontario midge (Chironomidae) AFP, which has intermediate antifreeze activity. Previously our group modelled the midge AFP based on its sequence characteristics with the crystal structure of Tenebrio molitor AFP as a template. The midge crystal structure at 1.9 Å-resolution is a close match to the modelled structure and shows a 10-residue repeated solenoid fold, with 8 disulfide-bonds stabilizing the coils and 7 Tyr pointing outward from one side of the solenoid structure as a potential ice-binding site. The second protein crystal structure is from Rhagium mordax, a longhorn beetle, solved at 2.05-Å resolution. This AFP is hyperactive and its crystal structure resembles that of the Rhagium inquisitor ortholog in having a β-solenoid fold with a wide, flat, ice-binding surface formed by four parallel rows of mainly Thr residues. The key difference between these structures is that the Rhagium mordax AFP has crystallized with its ice-binding site exposed to solvent providing the opportunity to see the organization of surface waters. In contrast the Rhagium inquisitor AFP crystallized with its ice-binding site making protein-protein contacts that obscure the surface water patterns. Supported by CIHR and the Canada Research Chair program

Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs.

Bacterial adhesins are modular cell-surface proteins that mediate adherence to other cells, surfaces, and ligands. The Antarctic bacterium Marinomonas primoryensis uses a 1.5-MDa adhesin comprising over 130 domains to position it on ice at the top of the water column for better access to oxygen and nutrients. We have reconstructed this 0.6-μm-long adhesin using a "dissect and build" structural biology approach and have established complementary roles for its five distinct regions. Domains in region I (RI) tether the adhesin to the type I secretion machinery in the periplasm of the bacterium and pass it through the outer membrane. RII comprises ~120 identical immunoglobulin-like β-sandwich domains that rigidify on binding Ca2+ to project the adhesion regions RIII and RIV into the medium. RIII contains ligand-binding domains that join diatoms and bacteria together in a mixed-species community on the underside of sea ice where incident light is maximal. RIV is the ice-binding domain, and the terminal RV domain contains several "repeats-in-toxin" motifs and a noncleavable signal sequence that target proteins for export via the type I secretion system. Similar structural architecture is present in the adhesins of many pathogenic bacteria and provides a guide to finding and blocking binding domains to weaken infectivity.

Our previously reported structures of calpain bound to its endogenous inhibitor calpastatin have motivated the use of aziridine aldehyde-mediated peptide macrocyclization toward the design of cyclic peptides and peptidomimetics as calpain inhibitors. Inspired by nature's hint that a β-turn loop within calpastatin forms a broad interaction around calpain's active site cysteine, we have constructed and tested a library of 45 peptidic compounds based on this loop sequence. Four molecules have shown reproducibly low micromolar inhibition of calpain-2. Further systematic sequence changes led to the development of probes that displayed increased potency and specificity of inhibition against calpain over other cysteine proteases. Calculated Ki values were in the low micromolar range, rivaling other peptidomimetic calpain inhibitors and presenting an improved selectivity profile against other therapeutically relevant proteases. Competitive and mixed inhibition against calpain-2 was observed, and an allosteric inhibition site on the enzyme was identified for a noncompetitive inhibitor.

Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed β-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the β-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the β-1 and β-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.

While ab initio modeling of protein structures is not routine, certain types of proteins are more straightforward to model than others. Proteins with short repetitive sequences typically exhibit repetitive structures. These repetitive sequences can be more amenable to modeling if some information is known about the predominant secondary structure or other key features of the protein sequence. We have successfully built models of a number of repetitive structures with novel folds using knowledge of the consensus sequence within the sequence repeat and an understanding of the likely secondary structures that these may adopt. Our methods for achieving this success are reviewed here.

Antifreeze proteins (AFPs) adsorb to ice through an extensive, flat, relatively hydrophobic surface. It has been suggested that this ice-binding site (IBS) organizes surface waters into an ice-like clathrate arrangement that matches and fuses to the quasi-liquid layer on the ice surface. On cooling, these waters join the ice lattice and freeze the AFP to its ligand. Evidence for the generality of this binding mechanism is limited because AFPs tend to crystallize with their IBS as a preferred protein-protein contact surface, which displaces some bound waters. Type III AFP is a 7 kDa globular protein with an IBS made up two adjacent surfaces. In the crystal structure of the most active isoform (QAE1), the part of the IBS that docks to the primary prism plane of ice is partially exposed to solvent and has clathrate waters present that match this plane of ice. The adjacent IBS, which matches the pyramidal plane of ice, is involved in protein-protein crystal contacts with few surface waters. Here we have changed the protein-protein contacts in the ice-binding region by crystallizing a fusion of QAE1 to maltose-binding protein. In this 1.9 Å structure, the IBS that fits the pyramidal plane of ice is exposed to solvent. By combining crystallography data with MD simulations, the surface waters on both sides of the IBS were revealed and match well with the target ice planes. The waters on the pyramidal plane IBS were loosely constrained, which might explain why other isoforms of type III AFP that lack the prism plane IBS are less active than QAE1. The AFP fusion crystallization method can potentially be used to force the exposure to solvent of the IBS on other AFPs to reveal the locations of key surface waters.

Significance A previously unidentified insect antifreeze protein has been characterized in a fly. It is present in the adult stage of midges that emerge from fresh water in the spring and need protection from occasional night frosts. The mature protein has 79 residues and is small and repetitive enough to be reliably modeled as a tightly wound solenoid structure. Each tandem repeat of 10 residues forms a coil of the solenoid that is cross-braced by a disulfide bond. This fold orients a row of seven stacked tyrosine side chains to make a flat surface, which is the predicted ice-binding site of the protein. This protein has no homologs in the database and has, therefore, recently evolved to protect its host from freezing.

When polypeptide chains fold into a protein, hydrophobic groups are compacted in the center with exclusion of water. We report the crystal structure of an alanine-rich antifreeze protein that retains ~400 waters in its core. The putative ice-binding residues of this dimeric, four-helix bundle protein point inwards and coordinate the interior waters into two intersecting polypentagonal networks. The bundle makes minimal protein contacts between helices, but is stabilized by anchoring to the semi-clathrate water monolayers through backbone carbonyl groups in the protein interior. The ordered waters extend outwards to the protein surface and likely are involved in ice binding. This protein fold supports both the anchored-clathrate water mechanism of antifreeze protein adsorption to ice and the water-expulsion mechanism of protein folding.

The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches.

Skeletal muscle-specific protein calpain-3 (CAPN3/p94) is a member of the calcium-dependent cysteine protease family. CAPN3 is indispensable for the maintenance and survival of functional skeletal muscles, and defects in the CAPN3 gene that cause loss of enzyme activity lead to the development of limb-girdle muscular dystrophy type 2A. The CAPN3 protease core (domains PC1 and 2) is particularly rich in these inactivating mutations. The whole enzyme, a homodimer of CAPN3, undergoes very rapid autoproteolysis. It has two insertion sequences, IS1 and IS2 not found in other calpains, and a long N-terminal region, NS. IS1 is present in the protease core. Previous biochemistry studies from our lab have shown that IS1 contributes to the instability of CAPN3 by acting as a readily cleavable internal pro-peptide that occupies the active site of the protease core. Here we report the 2.3Å resolution crystal structure of CAPN3 (ΔNS, C129S) protease core with Ca2+ ions present. Although the architecture of this protease core is similar to those of the CAPN1 and CAPN2, with two Ca2+ bound, the crystal structure of the CAPN3 core exhibits several novel features. The N-terminal anchor is more flexible than that of CAPN1 and CAPN2 even with the N-terminal 45 residues removed. Residues in the mutation-rich region of PC1 (207 to 234) form van der Waals and H-bond interactions with residues of both IS1 and helix10. The active site is blocked by part of IS1 that forms H-bonds through the OH groups of Tyr274 and Tyr322 to Trp360 from the active site, as well as a hydrophobic interaction between Tyr274 of IS1 to His334 of the catalytic triad. This aromatic stacking will hinder substrates and inhibitors from entering the active site. The crystal structure further confirms that IS1 is a critical region governing CAPN3 activation and the binding of substrates and inhibitors. Supported by funds from CIHR and the CRC program.

•PD150606 and LSEAL were thought to inhibit calpain by binding to PEF domains.•We tested inhibition of calpain and the protease core (no PEF domains present).•We find that PD150606 inhibits both full-length calpain and the protease core alone.•We do not find any inhibition of calpain by LSEAL in vitro.•PD150606 must inhibit calpain through binding to a site within the protease core.

Calpains are Ca2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca2+ signaling in the cell. The major isoforms of this enzyme family, calpain‐1 and calpain‐2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C‐terminal penta‐EF hand (PEF) domains. Calpain‐3, or p94, is a skeletal muscle‐specific isoform that is genetically linked to limb‐girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain‐3 support the suggestion that full‐length calpain‐3 exists as a homodimer. Here, we report the crystallization of calpain‐3′s PEF domain and its crystal structure in the presence of Ca2+, which provides evidence for the homodimer architecture of calpain‐3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain‐3 PEF domain contains a Ca2+ bound at the EF5‐hand used for homodimer association. Three of the four Ca2+‐binding EF‐hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain‐3 large subunit were to try to pair with a calpain small subunit.DatabaseStructural data are available in the Protein Data Bank database under accession number 4OKH.Structured digital abstractCalpain-3 and Calpain-3 bind by x-ray crystallography (View interaction)

A Ca2+‐dependent 1.5‐MDa antifreeze protein present in an Antarctic Gram‐negative bacterium, Marinomonas primoryensis (MpAFP), has recently been reassessed as an ice‐binding adhesin. The non‐ice‐binding region II (RII), one of five distinct domains in MpAFP, constitutes ~ 90% of the protein. RII consists of ~ 120 tandem copies of an identical 104‐residue sequence. We used the Protein Homology/analogy Recognition Engine server to define the boundaries of a single 104‐residue RII construct (RII monomer). CD demonstrated that Ca2+ is required for RII monomer folding, and that the monomer is fully structured at a Ca2+/protein molar ratio of 10 : 1. The crystal structure of the RII monomer was solved to a resolution of 1.35 Å by single‐wavelength anomalous dispersion and molecular replacement methods with Ca2+ as the heavy atom to obtain phase information. The RII monomer folds as a Ca2+‐bound immunoglobulin‐like β‐sandwich. Ca2+ ions are coordinated at the interfaces between each RII monomer and its symmetry‐related molecules, suggesting that these ions may be involved in the stabilization of the tandemly repeated RII. We hypothesize that > 600 Ca2+ ions help to rigidify the chain of 104‐residue repeats in order to project the ice‐binding domain of MpAFP away from the bacterial cell surface. The proposed role of RII is to help the strictly aerobic bacterium bind surface ice in an Antarctic lake for better access to oxygen and nutrients. This work may give insights into other bacterial proteins that resemble MpAFP, especially those of the large repeats‐in‐toxin family that have been characterized as adhesins exported via the type I secretion pathway.DatabaseStructural data are available in the Protein Data Bank under the accession numbers 4KDW (P1 structure) and 4KDV (P21 structure).

Force production by kinesins has been linked to structural rearrangements of the N and C termini of their motor domain upon nucleotide binding. In recent crystal structures, the Kar3-associated protein Vik1 shows unexpected homology to these conformational states even though it lacks a nucleotide-binding site. This conservation infers a degree of commonality in the function of the N- and C-terminal regions during the mechanochemical cycle of all kinesins and kinesin-related proteins. We tested this inference by examining the functional effects on Kar3Vik1 of mutating or deleting residues in Vik1 that are involved in stabilizing the C terminus against the core and N terminus of the Vik1 motor homology domain (MHD). Point mutations at two moderately conserved residues near the Vik1 C terminus impaired microtubule gliding and microtubule-stimulated ATP turnover by Kar3Vik1. Deletion of the seven C-terminal residues inhibited Kar3Vik1 motility much more drastically. Interestingly, none of the point mutants seemed to perturb the ability of Kar3Vik1 to bind microtubules, whereas the C-terminal truncation mutant did. Molecular dynamics simulations of these C-terminal mutants showed distinct root mean square fluctuations in the N-terminal region of the Vik1 MHD that connects it to Kar3. Here, the degree of motion in the N-terminal portion of Vik1 highly correlated with that in the C terminus. These observations suggest that the N and C termini of the Vik1 MHD form a discrete folding motif that is part of a communication pathway to the nucleotide-binding site of Kar3.