Adhesin domains responsible for binding bacteria to surfaces they colonize project outwards from companion split domains.

Bacterial adhesins attach their hosts to surfaces that the bacteria will colonize. This surface adhesion occurs through specific ligand-binding domains located towards the distal end of the long adhesin molecules. However, recognizing which of the many adhesin domains are structural and which are ligand binding has been difficult up to now. Here we have used the protein structure modeling program AlphaFold2 to predict structures for these giant 0.2- to 1.5-megadalton proteins. Crystal structures previously solved for several adhesin regions are in good agreement with the models. Whereas most adhesin domains are linked in a linear fashion through their N- and C-terminal ends, ligand-binding domains can be recognized by budding out from a companion core domain so that their ligand-binding sites are projected away from the axis of the adhesin for maximal exposure to their targets. These companion domains are "split" in their continuity by projecting the ligand-binding domain outwards. The "split domains" are mostly ß-sandwich extender modules, but other domains like a ß-solenoid can serve the same function. Bioinformatic analyses of Gram-negative bacterial sequences revealed wide variety ligand-binding domains are used in their Repeats-in-Toxin adhesins. The ligands for many of these domains have yet to be identified but known ligands include various cell-surface glycans, proteins, and even ice. Recognizing the ligands to which the adhesins bind could lead to ways of blocking colonization by bacterial pathogens. Engineering different ligand-binding domains into an adhesin has the potential to change the surfaces to which bacteria bind.

Horizontal Gene Transfer in Vertebrates: A Fishy Tale.

The recent assembly of the herring genome suggests this fish acquired its antifreeze protein gene by horizontal transfer and then passed a copy on to the smelt. The direction of gene transfer is confirmed by some accompanying transposable elements and by the breakage of gene synteny.

Chilling injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition.

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at -6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at -6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.

Mutations in the V-ATPase Assembly Factor VMA21 Cause a Congenital Disorder of Glycosylation With Autophagic Liver Disease.

BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.

Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis.

Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified 'splat' method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.

Carrot 'antifreeze' protein has an irregular ice-binding site that confers weak freezing point depression but strong inhibition of ice recrystallization.

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.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma- phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017.

High-capacity ice-recrystallization endpoint assay employing superhydrophobic coatings that is equivalent to the 'splat' assay.

We have developed an ice recrystallization inhibition (IRI) assay system that allows the side-by-side comparison of up to a dozen samples treated in an identical manner. This system is ideal for determining, by serial dilution, the IRI 'endpoint' where the concentration of a sample is reached that can no longer inhibit recrystallization. Samples can be an order of magnitude smaller in volume (<1 µL) than those used for the conventional 'splat' assay. The samples are pipetted into wells cut out of a superhydrophobic coating on sapphire slides that are covered with a second slide and then snap-frozen in liquid nitrogen. Sapphire is greatly superior to glass in its ability to cool quickly without cracking. As a consequence, the samples freeze evenly as a multi-crystalline mass. The ice grain size is slightly larger than that obtained by the 'splat' assay but can be followed sufficiently well to assess IRI activity by changes in mean grain boundary size. The slides can be washed in detergent and reused with no carryover of IRI activity even from the highest protein concentrations.

Flies expand the repertoire of protein structures that bind ice.

An antifreeze protein (AFP) with no known homologs has been identified in Lake Ontario midges (Chironomidae). The midge AFP is expressed as a family of isoforms at low levels in adults, which emerge from fresh water in spring before the threat of freezing temperatures has passed. The 9.1-kDa major isoform derived from a preproprotein precursor is glycosylated and has a 10-residue tandem repeating sequence xxCxGxYCxG, with regularly spaced cysteines, glycines, and tyrosines comprising one-half its 79 residues. Modeling and molecular dynamics predict a tightly wound left-handed solenoid fold in which the cysteines form a disulfide core to brace each of the eight 10-residue coils. The solenoid is reinforced by intrachain hydrogen bonds, side-chain salt bridges, and a row of seven stacked tyrosines on the hydrophobic side that forms the putative ice-binding site. A disulfide core is also a feature of the similar-sized beetle AFP that is a ß-helix with seven 12-residue coils and a comparable circular dichroism spectrum. The midge and beetle AFPs are not homologous and their ice-binding sites are radically different, with the latter comprising two parallel arrays of outward-pointing threonines. However, their structural similarities is an amazing example of convergent evolution in different orders of insects to cope with change to a colder climate and provide confirmation about the physical features needed for a protein to bind ice.

Fish antifreeze protein origin in sculpins by frameshifting within a duplicated housekeeping gene.

Antifreeze proteins (AFPs) are found in a variety of marine cold-water fishes where they prevent freezing by binding to nascent ice crystals. Their diversity (types I, II, III and antifreeze glycoproteins), as well as their scattered taxonomic distribution hint at their complex evolutionary history. In particular, type I AFPs appear to have arisen in response to the Late Cenozoic Ice Age that began ~ 34 million years ago via convergence in four different groups of fish that diverged from lineages lacking this AFP. The progenitor of the alanine-rich α-helical type I AFPs of sculpins has now been identified as lunapark, an integral membrane protein of the endoplasmic reticulum. Following gene duplication and loss of all but three of the 15 exons, the final exon, which encoded a glutamate- and glutamine-rich segment, was converted to an alanine-rich sequence by a combination of frameshifting and mutation. Subsequent gene duplications produced numerous isoforms falling into four distinct groups. The origin of the flounder type I AFP is quite different. Here, a small segment from the original antiviral protein gene was amplified and the rest of the coding sequence was lost, while the gene structure was largely retained. The independent origins of type I AFPs with up to 83% sequence identity in flounder and sculpin demonstrate strong convergent selection at the level of protein sequence for alanine-rich single alpha helices that bind to ice. Recent acquisition of these AFPs has allowed sculpins to occupy icy seawater niches with reduced competition and predation from other teleost species.

Superheating of ice crystals in antifreeze protein solutions.

It has been argued that for antifreeze proteins (AFPs) to stop ice crystal growth, they must irreversibly bind to the ice surface. Surface-adsorbed AFPs should also prevent ice from melting, but to date this has been demonstrated only in a qualitative manner. Here we present the first quantitative measurements of superheating of ice in AFP solutions. Superheated ice crystals were stable for hours above their equilibrium melting point, and the maximum superheating obtained was 0.44 degrees C. When melting commenced in this superheated regime, rapid melting of the crystals from a point on the surface was observed. This increase in melting temperature was more appreciable for hyperactive AFPs compared to the AFPs with moderate antifreeze activity. For each of the AFP solutions that exhibited superheating, the enhancement of the melting temperature was far smaller than the depression of the freezing temperature. The present findings clearly show that AFPs adsorb to ice surfaces as part of their mechanism of action, and this absorption leads to protection of ice against melting as well as freezing.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period.

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Smelt was the likely beneficiary of an antifreeze gene laterally transferred between fishes.

BACKGROUND: Type II antifreeze protein (AFP) from the rainbow smelt, Osmerus mordax, is a calcium-dependent C-type lectin homolog, similar to the AFPs from herring and sea raven. While C-type lectins are ubiquitous, type II AFPs are only found in a few species in three widely separated branches of teleost fishes. Furthermore, several other non-homologous AFPs are found in intervening species. We have previously postulated that this sporadic distribution has resulted from lateral gene transfer. The alternative hypothesis, that the AFP evolved from a lectin present in a shared ancestor and that this gene was lost in most species, is not favored because both the exon and intron sequences are highly conserved. RESULTS: Here we have sequenced and annotated a 160 kb smelt BAC clone containing a centrally-located AFP gene along with 14 other genes. Quantitative PCR indicates that there is but a single copy of this gene within the smelt genome, which is atypical for fish AFP genes. The corresponding syntenic region has been identified and searched in a number of other species and found to be devoid of lectin or AFP sequences. Unlike the introns of the AFP gene, the intronic sequences of the flanking genes are not conserved between species. As well, the rate and pattern of mutation in the AFP gene are radically different from those seen in other smelt and herring genes. CONCLUSIONS: These results provide stand-alone support for an example of lateral gene transfer between vertebrate species. They should further inform the debate about genetically modified organisms by showing that gene transfer between 'higher' eukaryotes can occur naturally. Analysis of the syntenic regions from several fishes strongly suggests that the smelt acquired the AFP gene from the herring.

Origin of an antifreeze protein gene in response to Cenozoic climate change.

Antifreeze proteins (AFPs) inhibit ice growth within fish and protect them from freezing in icy seawater. Alanine-rich, alpha-helical AFPs (type I) have independently (convergently) evolved in four branches of fishes, one of which is a subsection of the righteye flounders. The origin of this gene family has been elucidated by sequencing two loci from a starry flounder, Platichthys stellatus, collected off Vancouver Island, British Columbia. The first locus had two alleles that demonstrated the plasticity of the AFP gene family, one encoding 33 AFPs and the other allele only four. In the closely related Pacific halibut, this locus encodes multiple Gig2 (antiviral) proteins, but in the starry flounder, the Gig2 genes were found at a second locus due to a lineage-specific duplication event. An ancestral Gig2 gave rise to a 3-kDa "skin" AFP isoform, encoding three Ala-rich 11-a.a. repeats, that is expressed in skin and other peripheral tissues. Subsequent gene duplications, followed by internal duplications of the 11 a.a. repeat and the gain of a signal sequence, gave rise to circulating AFP isoforms. One of these, the "hyperactive" 32-kDa Maxi likely underwent a contraction to a shorter 3.3-kDa "liver" isoform. Present day starry flounders found in Pacific Rim coastal waters from California to Alaska show a positive correlation between latitude and AFP gene dosage, with the shorter allele being more prevalent at lower latitudes. This study conclusively demonstrates that the flounder AFP arose from the Gig2 gene, so it is evolutionarily unrelated to the three other classes of type I AFPs from non-flounders. Additionally, this gene arose and underwent amplification coincident with the onset of ocean cooling during the Cenozoic ice ages.

The Thr- and Ala-rich hyperactive antifreeze protein from inchworm folds as a flat silk-like ß-helix.

Inchworm larvae of the pale beauty geometer moth, Campaea perlata, exhibit strong (6.4 °C) freezing point depression activity, indicating the presence of hyperactive antifreeze proteins (AFPs). We have purified two novel Thr- and Ala-rich AFPs from the larvae as small (∼3.5 kDa) and large (∼8.3 kDa) variants and have cloned the cDNA sequences encoding both. They have no homology to known sequences in current BLAST databases. However, these proteins and the newly characterized AFP from the Rhagium inquisitor beetle both contain stretches rich in alternating Thr and Ala residues. On the basis of these repeats, as well as the discontinuities between them, a detailed structural model is proposed for the 8.3 kDa variant. This 88-residue protein is organized into an extended parallel-stranded ß-helix with seven strands connected by classic ß-turns. The alternating ß-strands form two ß-sheets with a thin core composed of interdigitating Ala and Ser residues, similar to the thin hydrophobic core proposed for some silks. The putative ice-binding face of the protein has a 4 × 5 regular array of Thr residues and is remarkably flat. In this regard, it resembles the nonhomologous Thr-rich AFPs from other moths and some beetles, which contain two longer rows of Thr in contrast to the five shorter rows in the inchworm protein. Like that of some other hyperactive AFPs, the spacing between these ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice.

Crystal waters on the nine polyproline type II helical bundle springtail antifreeze protein from Granisotoma rainieri match the ice lattice.

A springtail (Collembola) identified as Granisotoma rainieri was collected from snow in Hokkaido, Japan, in late winter when nighttime temperatures were below zero. Extracts of these arthropods showed antifreeze activity by shaping ice crystals and stopping their growth. The glycine-rich proteins responsible for this freezing point depression were isolated by ice-affinity purification and had principal masses of ~ 6.9 and 9.6 kDa. We identified a transcript for a 9.6-kDa component and produced it as a His-tagged recombinant protein for structural analysis. Its crystal structure was solved to a resolution of 1.21 Å and revealed a polyproline type II helical bundle, similar to the six-helix Hypogastrura harveyi AFP, but with nine helices organized into two layers held together by an extensive network of hydrogen bonds. One of the layers is flat, regular, and hydrophobic and likely serves as the ice-binding side. Although this surface makes close protein-protein contacts with its symmetry mate in the crystal, it has bound chains of waters present that resemble those on the basal and primary prism planes of ice. Molecular dynamic simulations indicate most of these crystal waters would preferentially occupy these sites if exposed to bulk solvent in the absence of the symmetry mate. These prepositioned waters lend further support to the ice-binding mechanism in which AFPs organize ice-like waters on one surface to adsorb to ice. DATABASES: Structural data are available in the Protein Data Bank under the accession number 7JJV. Transcript data are available in GenBank under accession numbers MT780727, MT780728, MT780729, MT780730, MT780731 and MT985982.

Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p.

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.

Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.

The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP.

Profiling of calpain activity with a series of FRET-based substrates.

Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.

Antifreeze protein complements cryoprotective dehydration in the freeze-avoiding springtail Megaphorura arctica.

The springtail, Megaphorura arctica, is freeze-avoiding and survives sub-zero temperatures by cryoprotective dehydration. At the onset of dehydration there is some supercooling of body fluids, and the danger of inoculative freezing, which would be lethal. To see if the springtails are protected by antifreeze proteins in this pre-equilibrium phase, we examined extracts from cold-acclimated M. arctica and recorded over 3 °C of freezing point depression. Proteins responsible for this antifreeze activity were isolated by ice affinity. They comprise isoforms ranging from 6.5 to 16.9 kDa, with an amino acid composition dominated by glycine (>35 mol%). Tryptic peptide sequences were used to identify the mRNA sequence coding for the smallest isoform. This antifreeze protein sequence has high similarity to one characterized in Hypogastrura harveyi, from a different springtail order. If these two antifreeze proteins are true homologs, we suggest their origin dates back to the Permian glaciations some 300 million years ago.