Segmentation and Comparative Modeling in an 8.6-Å Cryo-EM Map of the Singapore Grouper Iridovirus.

SGIV, or Singapore grouper iridovirus, is a large double-stranded DNA virus, reaching a diameter of 220 nm and packaging a genome of 140 kb. We present a 3D cryoelectron microscopy (cryo-EM) icosahedral reconstruction of SGIV determined at 8.6-Å resolution. It reveals several layers including a T = 247 icosahedral outer coat, anchor proteins, a lipid bilayer, and the encapsidated DNA. A new segmentation tool, iSeg, was applied to extract these layers from the reconstructed map. The outer coat was further segmented into major and minor capsid proteins. None of the proteins extracted by segmentation have known atomic structures. We generated models for the major coat protein using three comparative modeling tools, and evaluated each model using the cryo-EM map. Our analysis reveals a new architecture in the Iridoviridae family of viruses. It shares similarities with others in the same family, e.g., Chilo iridescent virus, but also shows new features of the major and minor capsid proteins.

Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody.

Hepatitis E virus (HEV), a non-enveloped, positive-sense, single-stranded RNA virus, is a major cause of enteric hepatitis. Classified into the family Hepeviridae, HEV comprises four genotypes (genotypes 1-4), which belong to a single serotype. We describe a monoclonal antibody (mAb), 8G12, which equally recognizes all four genotypes of HEV, with ∼ 2.53-3.45 nM binding affinity. The mAb 8G12 has a protective, neutralizing capacity, which can significantly block virus infection in host cells. Animal studies with genotypes 1, 3 and 4 confirmed the cross-genotype neutralizing capacity of 8G12 and its effective prevention of hepatitis E disease. The complex crystal structures of 8G12 with the HEV E2s domain (the most protruded region of the virus capsid) of the abundant genotypes 1 and 4 were determined at 4.0 and 2.3 Šresolution, respectively. These structures revealed that 8G12 recognizes both genotypes through the epitopes in the E2s dimerization region. Structure-based mutagenesis and cell-model assays with virus-like particles identified several conserved residues (Glu549, Lys554 and Gly591) that are essential for 8G12 neutralization. Moreover, the epitope of 8G12 is identified as a key epitope involved in virus-host interactions. These findings will help develop a common strategy for the prevention of the most abundant form of HEV infection.

Quantitative study of proteomic alterations in a Zebrafish (danio rerio) cell line infected with the Singapore Grouper Iridovirus (SGIV).

We demonstrate, for the first time, that Singapore Grouper Iridovirus (SGIV) can successfully infect a Zebrafish cell line. Combined with the recent availability of the complete zebrafish (danio rerio) genome, this provides an opportunity to investigate virus-host interactions at the molecular level. Using iTRAQ labeling and two-dimensional LC/MS/MS quantitative proteomics, 157 zebrafish proteins exhibiting significant alterations in expression levels following SGIV infection were identified. Gene ontology analysis revealed that SGIV controls a wide aspect of zebrafish host machinery to ensure replication and propagation. In order to probe the mechanism underlying SGIV infection in Zebrafish cells, we used an anti-sense morpholino to knockdown orf86r, an immediate early viral gene that encodes the SGIV protein ORF86R. The expression profile of certain host proteins involved in replication was altered upon knockdown. In particular, expression of CNOT, a non-enzymatic subunit of the CCR4-NOT transcription complex was markedly affected. Taken together, these findings provide a new insight on the function of the essential viral protein ORF86R. Our results show that Singapore Grouper Iridovirus infection of a Zebrafish cell line is a useful new tool to study virus-host interactions.

Structure of a novel shoulder-to-shoulder p24 dimer in complex with the broad-spectrum antibody A10F9 and its implication in capsid assembly.

Mature HIV-1 viral particles assemble as a fullerene configuration comprising p24 capsid hexamers, pentamers and dimers. In this paper, we report the X-ray crystal structures of the p24 protein from natural HIV-1 strain (BMJ4) in complex with Fab A10F9, which recognizes a conserved epitope in the C-terminal domain of the BMJ4 p24 protein. Our structures reveal a novel shoulder-to-shoulder p24 dimerization mode that is mediated by an S-S bridge at C177. Consistent with these structures, the shoulder-to-shoulder dimer that was obtained from the BMJ4 strain was also observed in p24 proteins from other strains by the introduction of a cysteine residue at position 177. The potential biological significance was further validated by the introduction of a C177A mutation in the BMJ4 strain, which then displays a low infectivity. Our data suggest that this novel shoulder-to-shoulder dimer interface trapped by this unique S-S bridge could represent a physiologically relevant mode of HIV-1 capsid assembly during virus maturation, although Cys residue itself may not be critical for HIV-I replication.

Membrane proteins of human fetal primitive nucleated red blood cells.

In humans, primitive fetal nucleated red blood cells (FNRBCs) are thought to be as vital for embryonic life as their counterpart, adult red blood cells (adult RBCs) are in later-gestation fetuses and adults. Unlike adult RBCs, the identity and functions of FNRBC proteins are poorly understood owing to a scarcity of FNRBCs for proteomic investigations. The study aimed to investigate membrane proteins of this unique cell type. We present here, the first report on the membrane proteome of human primitive FNRBCs investigated by two-dimensional liquid chromatography coupled with mass-spectrometry (2D-LCMS/MS) and bioinformatics analysis. A total of 273 proteins were identified, of which 133 (48.7%) were membrane proteins. We compared our data with membrane proteins of adult RBCs to identify common, and unique, surface membrane proteins. Twelve plasma membrane proteins with transmembrane domains and eight proteins with transmembrane domains but without known sub-cellular location were identified as unique-to-FNRBCs. Except for the transferrin receptor, all other 19 unique-to-FNRBC membrane proteins have never been described in RBCs. Reverse-transcriptase PCR (RT-PCR) and immunocytochemistry validated the 2D-LCMS/MS data. Our findings provide potential surface antigens for separation of primitive FNRBCs from maternal blood for noninvasive prenatal diagnosis, and to understand the biology of these rare cells.

Novel histone H3 binding protein ORF158L from the Singapore grouper iridovirus.

Singapore grouper iridovirus (SGIV), a major pathogen of concern for grouper aquaculture, has a double-stranded DNA (dsDNA) genome with 162 predicted open reading frames, for which a total of 62 SGIV proteins have been identified. One of these, ORF158L, bears no sequence homology to any other known protein. Knockdown of orf158L using antisense morpholino oligonucleotides resulted in a significant decrease in virus yield in grouper embryonic cells. ORF158L was observed in nuclei and virus assembly centers of virus-infected cells. This observation led us to study the structure and function of ORF158L. The crystal structure determined at 2.2-Å resolution reveals that ORF158L partially exhibits a structural resemblance to the histone binding region of antisilencing factor 1 (Asf1), a histone H3/H4 chaperon, despite the fact that there is no significant sequence identity between the two proteins. Interactions of ORF158L with the histone H3/H4 complex and H3 were demonstrated by isothermal titration calorimetry (ITC) experiments. Subsequently, the results of ITC studies on structure-based mutants of ORF158L suggested Arg67 and Ala93 were key residues for histone H3 interactions. Moreover, a combination of approaches of ORF158L knockdown and isobaric tags/mass spectrometry for relative and absolute quantifications (iTRAQ) revealed that ORF158L may be involved in both the regulation and the expression of histone H3 and H3 methylation. Our present studies suggest that ORF158L may function as a histone H3 chaperon, enabling it to control host cellular gene expression and to facilitate viral replication.

Structural basis for the neutralization and genotype specificity of hepatitis E virus.

Hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water, and is characterized by jaundice and flu-like aches and pains. To date, no vaccines are commercially available to prevent the disease caused by HEV. Previously, we showed that a monoclonal antibody, 8C11, specifically recognizes a neutralizing conformational epitope on HEV genotype I. The antibody 8C11 blocks the virus-like particle from binding to and penetrating the host cell. Here, we report the complex crystal structure of 8C11 Fab with HEV E2s(I) domain at 1.9 Å resolution. The 8C11 epitopes on E2s(I) were identified at Asp(496)-Thr(499), Val(510)-Leu(514), and Asn(573)-Arg(578). Mutations and cell-model assays identified Arg(512) as the most crucial residue for 8C11 interaction with and neutralization of HEV. Interestingly, 8C11 specifically neutralizes HEV genotype I, but not the other genotypes. Because HEV type I and IV are the most abundant genotypes, to understand this specificity further we determined the structure of E2s(IV) at 1.79 Å resolution and an E2s(IV) complex with 8C11 model was generated. The comparison between the 8C11 complexes with type I and IV revealed the key residues that distinguish these two genotypes. Of particular interest, the residue at amino acid position 497 at the 8C11 epitope region of E2s is distinct among these two genotypes. Swapping this residue from one genotype to another inversed the 8C11 reactivity, demonstrating the essential role played by amino acid 497 in the genotype recognition. These studies may lead to the development of antibody-based drugs for the specific treatment against HEV.

DNA condensates organized by the capsid protein VP15 in White Spot Syndrome Virus.

The White Spot Syndrome Virus (WSSV) has a large circular double-stranded DNA genome of around 300kb and it replicates in the nucleus of the host cells. The machinery of how the viral DNA is packaged has been remained unclear. VP15, a highly basic protein, is one of the major capsid proteins found in the virus. Previously, it was shown to be a DNA binding protein and was hypothesized to participate in the viral DNA packaging process. Using Atomic Force Microscopy imaging, we show that the viral DNA is associated with a (or more) capsid proteins. The organized viral DNA qualitatively resembles the conformations of VP15 induced DNA condensates in vitro. Furthermore, single-DNA manipulation experiments revealed that VP15 is able to condense single DNA against forces of a few pico Newtons. Our results suggest that VP15 may aid in the viral DNA packaging process by directly condensing DNA.

Hypoxia-like effect of cobalt chromium alloy micro particles on fibroblasts in vitro.

Periprosthetic osteolysis leading to asceptic loosening remains the primary cause of failure of joint replacement. Although many inflammatory cell types have been implicated, the exact pathomechanisms of asceptic loosening have not been delineated. In the present study we have adopted a proteomic approach to elucidate the initial signals that are expressed to particulate material, using an in vitro cell culture system. Human lung fibroblasts MRC-5 were cultured with Cobalt Chromium (CoCr ASTM F-75, 1-7 µm) particles. Cells were harvested after 72 h incubation and total cellular proteins extracted for downstream analysis via 2D Gel Electrophoresis and tandem mass spectrometry using MALDI-TOF-TOF-MS. Thirteen protein spots showed greater than twofold increase, following 72 h incubation of fibroblast with CoCr particles. Four of these proteins were identified by tandem mass spectrometry. These were Annexin II, Pyruvate kinase, Triose phosphate isomerase, and N-myc downstream regulated gene 1 protein. Cobalt is a hypoxia mimicking agent and N-myc downstream regulated gene 1 protein, Triose phosphate isomerase, Pyruvate kinase, and Annexin II are important hypoxia regulated gene products that are found to be over expressed in cellular oxidative stress response. Our data indicates that exposure of fibroblast to CoCr alloy induces the transition of these cells into a hypoxia like state and oxidative stress even in normoxic culture conditions. The study reflects the possibility of the presence of a hypoxic environment in the periprosthetic tissue surrounding metallic implants.

Lipidomic study of intracellular Singapore grouper iridovirus.

Singapore grouper iridoviruses (SGIV) infected grouper cells release few enveloped extracellular viruses by budding and many unenveloped intracellular viruses following cell lysis. The lipid composition and function of such unenveloped intracellular viruses remain unknown. Detergent treatment of the intracellular viruses triggered the loss of viral lipids, capsid proteins and infectivity. Enzymatic digestion of the viral lipids with phospholipases and sphingomyelinase retained the viral capsid proteins but reduced infectivity. Over 220 lipid species were identified and quantified from the viruses and its producer cells by electrospray ionization mass spectrometry. Ten caspid proteins that dissociated from the viruses following the detergent treatments were identified by MALDI-TOF/TOF-MS/MS. Five of them were demonstrated to be lipid-binding proteins. This is the first research detailing the lipidome and lipid-protein interactions of an unenveloped virus. The identified lipid species and lipid-binding proteins will facilitate further studies of the viral assembly, egress and entry.

Dimerization of hepatitis E virus capsid protein E2s domain is essential for virus-host interaction.

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV-host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.

iTRAQ analysis of Singapore grouper iridovirus infection in a grouper embryonic cell line.

We report, here, the first proteomics study of a grouper embryonic cell line (GEC) infected by Singapore grouper iridovirus (SGIV). The differential proteomes of GEC with and without viral infection were studied and quantified with iTRAQ labelling followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Forty-nine viral proteins were recognized, of which 11 were identified for the first time. Moreover, 743 host proteins were revealed and classified into 218 unique protein groups. Fourteen host proteins were upregulated and five host proteins were downregulated upon viral infection. The iTRAQ analysis of SGIV infection in GEC provides an insight to viral and host gene products at the protein level. This should facilitate further study and the understanding of virus-host interactions, molecular mechanisms of viral infection and pathogenesis.

ORF018R, a highly abundant virion protein from Singapore grouper iridovirus, is involved in serine/threonine phosphorylation and virion assembly.

Iridovirus is an important pathogen causing serious diseases among wild, cultured and ornamental fish. Previous studies have shown that Singapore grouper iridovirus (SGIV) contains 162 open reading frames (ORFs) from which 51 viral proteins have been confirmed by proteomics studies. ORF018R, which is conserved among vertebrate iridoviruses, is an abundant virion protein identified from SGIV. Here, immunofluorescence staining showed that ORF018R occurred at high abundance throughout SGIV-infected cells. The function of ORF018R was explored using antisense morpholino oligonucleotides (asMOs). Knockdown of ORF018R expression resulted in a reduction in the expression of viral late genes, distortion of viral particle assembly and inhibition of SGIV infection in grouper embryonic cells. Western blotting with phosphoserine-specific antibody indicated that serine phosphorylation was significantly enhanced for proteins of molecular masss 17-32 kDa by SDS-PAGE when ORF018R expression was eliminated. These proteins were analysed further by two-dimensional gel electrophoresis, and numerous protein spots were found to shift to a lower pI and higher molecular mass as a result of the loss of ORF018R function. Five proteins with enhanced phosphorylation were identified by matrix-assisted laser desorption/ionization time-of-flight (TOF)-TOF mass spectrometry, including three viral proteins: ORF049L (dUTPase), ORF075R and ORF086R, and two host proteins: subunit 12 of eukaryotic translation factor 3 and natural killer enhancing factor. These findings suggest that ORF018R is involved in serine/threonine phosphorylation in SGIV-infected late-stage cells and plays an important role in expression of viral late genes and virion assembly.

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Differential expression of novel tyrosine kinase substrates during breast cancer development.

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes in the MCF10AT model of breast cancer progression using a combination of phosphotyrosyl affinity enrichment, iTRAQ technology, and LC-MS/MS. Using complementary MALDI- and ESI-based mass spectrometry, 57 unique proteins comprising tyrosine kinases, phosphatases, and other signaling proteins were detected to undergo differential phosphorylation during disease progression. Seven of these proteins (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, and RPS2) were validated to be novel tyrosine kinase substrates. SPAG9, TOLLIP, WBP2, and NSFL1C were further proven to be authentic targets of epidermal growth factor signaling and Iressa (gefitinib). A closer examination revealed that the expression of SLC4A7, a bicarbonate transporter, was down-regulated in 64% of the 25 matched normal and tumor clinical samples. The expression of TOLLIP in clinical breast cancers was heterogeneous with 25% showing higher expression in tumor compared with normal tissues and 35% showing the reverse trend. Preliminary studies on SPAG9, on the other hand, did not show differential expression between normal and diseased states. This is the first time SLC4A7 and TOLLIP have been discovered as novel tyrosine kinase substrates that are also associated with human cancer development. Future molecular and functional studies will provide novel insights into the roles of TOLLIP and SLC4A7 in the molecular etiology of breast cancer.

White spot syndrome virus proteins and differentially expressed host proteins identified in shrimp epithelium by shotgun proteomics and cleavable isotope-coded affinity tag.

Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, > or =20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.

Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus.

White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 A resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 A. SeMet-labelled VP28 crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 A, and diffracts to 2.0 A resolution.

Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells.

With the completion of the human genome project, analysis of enriched phosphotyrosyl proteins from epidermal growth factor (EGF)-induced phosphotyrosine proteome permits the identification of novel downstream substrates of the EGF receptor (EGFR). Using cICAT-based LC-MS/MS method, we identified and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR. In addition, EGFR activity was shown to be necessary for EGF-induced localization of Endofin, an FYVE domain-containing protein regulated by phosphoinositol lipid and engaged in endosome-mediated receptor modulation. Although several groups have conducted phosphoproteomics of EGF signaling in recent years, our study is the first to identify and validate Endofin, DCBLD2, and KIAA0582 as part of a complex EGF phosphotyrosine signaling network. These novel data will provide new insights into the complex EGF signaling and may have implications on target-directed cancer therapeutics.

Shotgun identification of the structural proteome of shrimp white spot syndrome virus and iTRAQ differentiation of envelope and nucleocapsid subproteomes.

White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomics studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using off-line coupling of an LC system with MALDI-TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach led to the identification of 45 viral proteins; 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomics strategy, was used to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we successfully identified 23 envelope proteins and six nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore the localization of an additional 12 envelope proteins and two nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high throughput viral protein localization determination. Altogether WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one identified by iTRAQ. The localization of 42 structural proteins was determined; 33 are envelope proteins, and nine are nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.

Structure and evolutionary origin of Ca(2+)-dependent herring type II antifreeze protein.

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca(2+)-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca(2+)-dependent) lectins with unique ice-binding features. The 1.7 A crystal structure of hAFP with bound Ca(2+) and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca(2+)-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca(2+) through the coordination with a water molecule of the ice lattice. This Ca(2+)-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.