Evolution of the lipocalin family as inferred from a protein sequence phylogeny

The lipocalins constitute a family of proteins that have been found in eubacteria and a variety of eukaryotic cells, where they play diverse physiological roles. It is the primary goal of this review to examine the patterns of change followed by lipocalins through their complex history, in order to stimulate scientists in the field to experimentally contrast our phylogeny-derived hypotheses. We reexamine our previous work on lipocalin phylogeny and update the phylogenetic analysis of the family. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency.     

A phylogenetic analysis of the lipocalin protein family

The lipocalins are a family of extracellular proteins that bind and transport small hydrophobic molecules. They are found in eubacteria and a great variety of eukaryotic cells, in which they play diverse physiological roles. We report here the detection of two new eukaryotic lipocalins and a phylogenetic analysis of 113 lipocalin family members performed with maximum-likelihood and parsimony methods on their amino acid sequences. Lipocalins segregate into 13 monophyletic clades, some of which are grouped in well-supported superclades. An examination of the G + C content of the bacterial lipocalin genes and the detection of four new conceptual lipocalins in other eubacterial species argue against a recent horizontal transfer as the origin of prokaryotic lipocalins. Therefore, we rooted our lipocalin tree using the clade containing the prokaryotic lipocalins. The topology of the rooted lipocalin tree is in general agreement with the currently accepted view of the organismal phylogeny of arthropods and chordates. The rooted tree allows us to assign polarity to character changes and suggests a plausible scenario for the evolution of important lipocalin properties. More recently evolved lipocalins tend to (1) show greater rates of amino acid substitutions, (2) have more flexible protein structures, (3) bind smaller hydrophobic ligands, and (4) increase the efficiency of their ligand-binding contacts. Finally, we found that the family of fatty-acid-binding proteins originated from the more derived lipocalins and therefore cannot be considered a sister group of the lipocalin family.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

The transthyretin-related protein: structural investigation of a novel protein family

The transthyretin-related protein (TRP) family comprises proteins predicted to be structurally related to the homotetrameric transport protein transthyretin (TTR). The function of TRPs is not yet fully established, but recent data suggest that they are involved in purine catabolism. We have determined the three-dimensional structure of the Escherichia coli TRP in two crystal forms; one at 1.65 A resolution in the presence of zinc, and the other at 2.1 A resolution in the presence of zinc and bromide. The structures revealed five zinc-ion-binding sites per monomer. Of these, the zinc ions bound at sites I and II are coordinated in tetrahedral geometries to the side chains of residues His9, His96, His98, Ser114, and three water molecules at the putative ligand-binding site. Of these four residues, His9, His98, and Ser114 are conserved. His9 and His98 bind the central zinc (site I) together with two water molecules. The side chain of His98 also binds to the zinc ion at site II. Bromide ions bind at site I only, replacing one of the water molecules coordinated to the zinc ion. The C-terminal four amino acid sequence motif Y-[RK]-G-[ST] constitutes the signature sequence of the TRP family. Two Tyr111 residues form direct hydrogen bonds to each other over the tetramer interface at the area, which in TTR constitutes the rear part of its thyroxine-binding channel. The putative substrate/ligand-binding channel of TRP is consequently shallower and broader than its counterpart in TTR.     

Evolutionary coupling of structural and functional sequence information in the intracellular lipid-binding protein family

We have mined the evolutionary record for the large family of intracellular lipid-binding proteins (iLBPs) by calculating the statistical coupling of residue variations in a multiple sequence alignment using methods developed by Ranganathan and coworkers (Lockless and Ranganathan, Science 1999:286;295-299). The 213 sequences analyzed have a wide range of ligand-binding functions as well as highly divergent phylogenetic origins, assuring broad sampling of sequence space. Emerging from this analysis were two major clusters of coupled residues, which when mapped onto the structure of a representative iLBP under study in our laboratory, cellular retinoic-acid binding protein I, are largely contiguous and provide useful points of comparison to available data for the folding of this protein. One cluster comprises a predominantly hydrophobic core away from the ligand-binding site and likely represents key structural information for the iLBP fold. The other cluster includes the portal region where ligand enters its binding site, regions of the ligand-binding cavity, and the region where the 10-stranded beta-barrel characteristic of this family closes (between strands 1' and 10). Linkages between these two clusters suggest that evolutionary pressures on this family constrain structural and functional sequence information in an interdependent fashion. The necessity of the structure to wrap around a hydrophobic ligand confounds the typical sequestration of hydrophobic side chains. Additionally, ligand entry and exit require these structures to have a capacity for specific conformational change during binding and release. We conclude that an essential and structurally apparent separation of local and global sequence information is conserved throughout the iLBP family.     

An intriguing member of the lipocalin protein family: alpha 1-microglobulin.

The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system.     

The pur protein family: Genetic and structural features in development and disease

The Pur proteins are an ancient family of sequence‐specific single‐stranded nucleic acid‐binding proteins. They bind a G‐rich element in either single‐ or double‐stranded nucleic acids and are capable of displacing the complementary C‐rich strand. Recently several reports have described Pur family member knockouts, mutations, and disease aberrations. Together with a recent crystal structure of Purα, these data reveal conserved structural features of these proteins that have been adapted to serve functions unique to higher eukaryotes. In humans Pur proteins are critical for myeloid cell development, muscle development, and brain development, including trafficking of mRNA to neuronal dendrites. Pur family members have been implicated in diseases as diverse as cancer, premature aging, and fragile‐X mental retardation syndrome. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.     

Mouse oncogene protein 24p3 is a member of the lipocalin protein family.

Rigorous new methods of protein sequence analysis have been applied to the lipocalins, a diverse family of ligand binding proteins. Using three conserved sequence motifs to search for similar patterns in a large sequence database, the size and composition of this protein family have been defined in an automatic and objective way. It has allowed the identification of an existing sequence, mouse 24p3 protein, as a lipocalin and the possible rejection of other putative members from this protein family. On the basis of this newly discovered homology, a possible function for mouse 24p3 protein is proposed.     

Predicting protein function from sequence and structural data

When a protein's function cannot be experimentally determined, it can often be inferred from sequence similarity. Should this process fail, analysis of the protein structure can provide functional clues or confirm tentative functional assignments inferred from the sequence. Many structure-based approaches exist (e.g. fold similarity, three-dimensional templates), but as no single method can be expected to be successful in all cases, a more prudent approach involves combining multiple methods. Several automated servers that integrate evidence from multiple sources have been released this year and particular improvements have been seen with methods utilizing the Gene Ontology functional annotation schema.     

The CATH database: an extended protein family resource for structural and functional genomics

The CATH database of protein domain structures (http://www.biochem.ucl.ac.uk/bsm/cath_new) currently contains 34 287 domain structures classified into 1383 superfamilies and 3285 sequence families. Each structural family is expanded with domain sequence relatives recruited from GenBank using a variety of efficient sequence search protocols and reliable thresholds. This extended resource, known as the CATH-protein family database (CATH-PFDB) contains a total of 310 000 domain sequences classified into 26 812 sequence families. New sequence search protocols have been designed, based on these intermediate sequence libraries, to allow more regular updating of the classification. Further developments include the adaptation of a recently developed method for rapid structure comparison, based on secondary structure matching, for domain boundary assignment. The philosophy behind CATHEDRAL is the recognition of recurrent folds already classified in CATH. Benchmarking of CATHEDRAL, using manually validated domain assignments, demonstrated that 43% of domains boundaries could be completely automatically assigned. This is an improvement on a previous consensus approach for which only 10-20% of domains could be reliably processed in a completely automated fashion. Since domain boundary assignment is a significant bottleneck in the classification of new structures, CATHEDRAL will also help to increase the frequency of CATH updates.     

The protein family of pyruvate:quinone oxidoreductases: Amino acid sequence conservation and taxonomic distribution

Pyruvate:quinone oxidoreductases (PQOs) catalyse the oxidative decarboxylation of pyruvate to acetate and concomitant reduction of quinone to quinol with the release of CO₂. They are thiamine pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) containing enzymes, which interact with the membrane in a monotopic way. PQOs are considered as part of alternatives to most recognized pyruvate catabolizing pathways, and little is known about their taxonomic distribution and structural/functional relationship.In this bioinformatics work we tackled these gaps in PQO knowledge. We used the KEGG database to identify PQO coding genes, performed a multiple sequence analysis which allowed us to study the amino acid conservation on these enzymes, and looked at their possible cellular function. We observed that PQOS are enzymes exclusively present in prokaryotes with most of the sequences identified in bacteria. Regarding the amino acid sequence conservation, we found that 75 amino acid residues (out of 570, on average) have a conservation over 90 %, and that the most conserved regions in the protein are observed around the TPP and FAD binding sites. We systematized the presence of conserved features involved in Mg²⁺, TPP and FAD binding, as well as residues directly linked to the catalytic mechanism. We also established the presence of a new motif named “HEH lock”, possibly involved in the dimerization process. The results here obtained for the PQO protein family contribute to a better understanding of the biochemistry of these respiratory enzymes.     

The transglutaminase family: an overview: Minireview article

Summary. The knowledge that very different processes such as normal and neoplastic cell growth, reproduction and death are dependent on the presence of adequate levels of transglutaminases (TGase: EC 2.3.2.13) and that they are capable of affecting the differentiation and proliferative capability of several cell types, has prompted a multitude of researchers to study these fascinating molecules. In the following overview we intend to summarize the currently known information on the biological significance of these enzymes.     

A structural overview of the ion channels of the TRPM family

The TRPM (transient receptor potential melastatin) family belongs to the superfamily of TRP cation channels. The TRPM subfamily is composed of eight members that are involved in diverse biological functions such as temperature sensing, inflammation, insulin secretion, and redox sensing. Since the first cloning of TRPM1 in 1998, tremendous progress has been made uncovering the function, structure, and pharmacology of this family. Complete structures of TRPM2, TRPM4, and TRPM8, as well as a partial structure of TRPM7, have been determined by cryo-EM, providing insights into their channel assembly, ion permeation, gating mechanisms, and structural pharmacology. Here we summarize the current knowledge about channel structure, emphasizing general features and principles of the structure of TRPM channels discovered since 2017. We also discuss some of the key unresolved issues in the field, including the molecular mechanisms underlying voltage and temperature dependence, as well as the functions of the TRPM channels’ C-terminal domains.     

A lipocalin sequence signature modulates cell survival

Lipocalins are β-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property.     

Subfamily I Treponema pallidum repeat protein family: sequence variation and immunity

A 12-membered Treponema pallidum repeat (Tpr) protein family has been identified in T. pallidum subsp. pallidum, the causative agent of syphilis. The subfamily I Tpr proteins (C, D, F, and I) possess conserved sequence at the N- and C-termini and central regions that differentiate the members. These proteins may be important in the immune response during syphilis infection and in protective immunity. Strong antibody responses have been observed toward some of the subfamily I Tpr proteins during infection with different syphilis isolates. Some sequence variation has also been identified in one subfamily I Tpr member, TprD, among T. pallidum subsp. pallidum isolates. In this study, we examined sequences in the remaining subfamily I Tpr proteins among strains. Both TprF and TprI were conserved among T. pallidum subsp. pallidum isolates.While some heterogeneity was identified in TprC. We further examined the immune response and protective capacity of TprF protein in this paper. We demonstrate that the N-terminal conserved region of the subfamily I Tpr proteins elicits strong antibody and T-cell responses during infection, and immunization with this region attenuates syphilitic lesion development upon infectious challenge.     

Thoroughly sampling sequence space: large-scale protein design of structural ensembles

Modeling the inherent flexibility of the protein backbone as part of computational protein design is necessary to capture the behavior of real proteins and is a prerequisite for the accurate exploration of protein sequence space. We present the results of a broad exploration of sequence space, with backbone flexibility, through a novel approach: large-scale protein design to structural ensembles. A distributed computing architecture has allowed us to generate hundreds of thousands of diverse sequences for a set of 253 naturally occurring proteins, allowing exciting insights into the nature of protein sequence space. Designing to a structural ensemble produces a much greater diversity of sequences than previous studies have reported, and homology searches using profiles derived from the designed sequences against the Protein Data Bank show that the relevance and quality of the sequences is not diminished. The designed sequences have greater overall diversity than corresponding natural sequence alignments, and no direct correlations are seen between the diversity of natural sequence alignments and the diversity of the corresponding designed sequences. For structures in the same fold, the sequence entropies of the designed sequences cluster together tightly. This tight clustering of sequence entropies within a fold and the separation of sequence entropy distributions for different folds suggest that the diversity of designed sequences is primarily determined by a structure's overall fold, and that the designability principle postulated from studies of simple models holds in real proteins. This has important implications for experimental protein design and engineering, as well as providing insight into protein evolution.     

Hinge Atlas: relating protein sequence to sites of structural flexibility

Background  

Relating features of protein sequences to structural hinges is important for identifying domain boundaries, understanding structure-function relationships, and designing flexibility into proteins. Efforts in this field have been hampered by the lack of a proper dataset for studying characteristics of hinges.     

Bioinformatics of granzymes: sequence comparison and structural studies on granzyme family by homology modeling

Cytotoxic lymphocytes (CTLs), the key players of cell mediated immunity, induce apoptosis by engaging death receptors or through exocytosis of cytolytic granules containing granzyme (proteases) and pore-forming protein (perforin). The crystal structure of granzyme B from human (B(h)) and rat (B(r)), as well as that of pro-granzyme K (K(h)) has been reported recently. In the present communication, we describe the homology modeling of granzyme family (in particular Gzm A(h), M(h), B(m), and C(m) from human and mouse) based on the crystal structural coordinates of trypsin, granzyme K (K(h)), and granzyme B (B(h)). These models have been used for establishing phylogenetic relationship as well as identifying characteristic features for designing specific inhibitors. The paper also highlights key residues at the S1, S2, and S2(') binding subsites in all granzyme, which may be involved in the structure-function relationship of this enzyme family. The predicted 3D homology models show a conserved two similar domain structure, i.e., an N-terminal domain and a C-terminal domain comprising predominantly of beta-sheet structure with a little alpha-helical content. Micro-heterogeneities have been observed in the vicinity of the active site in all granzymes as compared to granzyme B(h). For example, in granzyme M(h), valine is present at the S1 subsite instead of arginine. Similarly differences at S2 (Leu-->Phe), S3 (Ser-->Gly), and S4 (Arg-->Asn) subsites are quite apparent and appear to hold the potential for selective designing of inhibitors for possible therapeutic applications. Furthermore, analysis of the electrostatic surface potential on the shape of granzyme-inhibitor binding groove reveals clear differences at the reactive site. Additionally the different posttranslational modification sites such as phosphorylation (e.g., in granzyme M Thr101, Ser109), myristoylation (Gly22, 117, and 131), and glycosylation (Ser160) have been identified, as very little is known about the functional significance of these modifications in the granzyme family. Thus, glycosylation at Ser160 in granzyme M may influence the net charge of the enzyme, resulting in altered substrate binding as compared to granzyme B. Also this modification may influence the rate of complexation and binding affinity with proteoglycans. These studies are expected to contribute towards the basic understanding of functional associations of the granzymes with other molecules and their possible role in apoptosis.