Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters. For the crenarchaeon Sulfolobus solfataricus P2, the repeats in the genome fall mainly into two closely related sequence families that are arranged in seven clusters containing a total of 441 repeats which constitute ca. 1% of the genome. The Sulfolobus conjugative plasmid pNOB8 contains a small cluster of six repeats that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also recognizes both main families of repeat sequences in S. solfataricus. The recombinant protein, expressed in Escherichia coli, showed the same binding properties to the SRSR repeat as the native one. The SSO454 protein exhibits a tripartite internal repeat structure which yields a good sequence match with a helix-turn-helix DNA-binding motif. Although this putative motif is shared by other archaeal proteins, orthologs of SSO454 were only detected in species within the Sulfolobus genus and in the closely related Acidianus genus. We infer that the genus-specific protein induces an opening of the structure at the center of each DNA repeat and thereby produces a binding site for another protein, possibly a more conserved one, in a process that may be essential for higher-order stucturing of the SRSR clusters.     

Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins

Seven imperfect repeats of a 40-amino acid cysteine-rich sequence constitute the ligand binding domain of the low density lipoprotein (LDL) receptor. To assess the contribution of each repeat, three site-directed mutations were made individually in each repeat: 1) deletion of the repeat, 2) substitution of a conserved isoleucine with aspartic acid, and 3) substitution of a conserved aspartic acid with tyrosine. cDNAs containing these mutations were transfected into simian COS cells and assayed for their ability to bind LDL, which contains a 500-kDa protein ligand (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains multiple copies of a 33-kDa ligand (apoE). The results showed that binding of the two ligands required different combinations of repeats. LDL binding required repeats 3-7; deletion of any one of these repeats markedly reduced LDL binding. In contrast, beta-migrating very low density lipoprotein binding was insensitive to the loss of any single repeat with the important exception of repeat 5, whose loss reduced binding by 60%. The same effects were obtained when each of the repeats was altered by either of the two substitution mutations. The current findings suggest that a multiplicity of cysteine-rich repeats may allow a single protein to bind several different protein ligands by employing different combinations of repeats.     

Mutational analysis of the ligand binding domain of the low density lipoprotein receptor

The ligand binding domain of the low density lipoprotein (LDL) receptor contains seven imperfect repeats of a 40-amino acid cysteine-rich sequence. Each repeat contains clustered negative charges that have been postulated as ligand-binding sites. The adjacent region of the protein, the growth factor homology region, contains three cysteine-rich repeats (A-C) whose sequence differs from those in the ligand binding domain. To dissect the contribution of these different cysteine-rich repeats to ligand binding, we used oligonucleotide-directed mutagenesis to alter expressible cDNAs for the human LDL receptor which were then introduced into monkey COS cells by transfection. We measured the ability of the mutant receptors to bind LDL, which contains a single protein ligand for the receptor (apoB-100), and beta-migrating very low density lipoprotein (beta-VLDL), which contains apoB-100 plus multiple copies of another ligand (apoE). The results show that repeat 1 is not required for binding of either ligand. Repeats 2 plus 3 and repeats 6 plus 7 are required for maximal binding of LDL, but not beta-VLDL. Repeat 5 is required for binding of both ligands. Repeat A in the growth factor homology region is required for binding of LDL, but not beta-VLDL. Repeat B is not required for ligand binding. These results support a model for the LDL receptor in which various repeats play additive roles in ligand binding, each repeat making a separate contribution to the binding event.     

Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus.

The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.     

Taxonomic distribution,repeats, and functions of the S1 domain‐containing proteins as members of the OB‐fold family

Proteins of the nucleic acid‐binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB‐fold in protein structures. Here, we have analyzed the superfamily of nucleic acid‐binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA‐binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA‐binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain‐containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB‐fold is a distinctive feature of S1 domain‐containing proteins. Proteins from the other families and superfamilies have either one OB‐fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain‐containing proteins. Proteins 2017; 85:602–613. © 2016 Wiley Periodicals, Inc.     

Expression,Purification and Structural Analysis of a Fibrinogen Receptor FbsA from <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis>

Streptococcus agalactiae is a leading cause of bacterial sepsis and meningitis in neonates. FbsA, a fibrinogen receptor of S. agalactiae is highly repetitive protein with each repeat containing 16 amino acids. The protein sequence of FbsA shows no homology to any known fibrinogen binding protein from other bacterial species, making it a unique fibrinogen receptor. FbsA is cloned, expressed in E. coli and purified. The recombinant protein shows a laddering pattern in SDS–PAGE gel because of its poor stability in solution. The instability of the protein is probably because of the presence Gln-Gly dipeptide in each repeat. The circular dichroism study of FbsA has shown that the protein is composed of alpha helices predominantly and random coils to a lesser extent, which agrees with the predicted secondary structure. Ab initio modeling of a single repeat shows that FbsA is made up of mainly alpha helix and the structural model of multiple repeats (3 or 4) suggests that the protein might adopt some form of a repeating helical structure and the overall conformation of the molecule might change depending on the number of repeats.     

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide,a bacterial cell wall fragment

Repeat proteins have recently emerged as especially well‐suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence‐structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand‐recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N‐ and C‐capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Using peptide arrays to define nuclear carrier binding sites on nucleoporins

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.     

Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats

The primary structure of the ribonuclease inhibitor from pig liver has been determined by amino acid sequence analysis. The N alpha-acetylated polypeptide chain of 456 amino acids consists of 15 homologous leucine-rich repeats, characterized by leucyl residues at constant positions. Two types of alternating repeats occur, 29 (A) and 28 (B) residues long. The degree of identity between repeats of a given type ranged from 25 to 60%. Only one deletion in the B-repeat was necessary to perfectly align the leucyl residues between the two repeats. Leucine-rich repeats have previously been found in four membrane-bound proteins and one extracellular protein, and their amphiphilic character suggested that they could be involved in membrane binding. Ribonuclease inhibitor is the first example of a cytoplasmic protein containing this type of repeat. It seems likely, therefore, that leucine-rich repeats can have functions other than forming membrane binding structures.     

Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase

The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC–RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

A plant gene encoding a Myb-like protein that binds telomeric GGTTTAG repeats in vitro

A gene (AtTRP1) encoding a telomeric repeat-binding protein has been isolated from Arabidopsis thaliana. AtTRP1 is a single copy gene located on chromosome 5 of A. thaliana. The protein AtTRP1 encoded by this gene is not only homologous to the Myb DNA-binding motifs of other telomere-binding proteins but also is similar to several initiator-binding proteins in plants. Gel retardation assay revealed that the 115 residues on the C terminus of this protein, including the Myb motif, are sufficient for binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does not form any complex at all with the DNA fragments carrying four or fewer GGTTTAG repeats. However, it forms a complex with the sequence (GGTTTAG)(8) more efficiently than with the sequence (GGTTTAG)(5). These data suggest that the minimum length of a telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and that the optimal AtTRP1 binding may require eight or more GGTTTAG repeats. It also implies that this protein AtTRP1 may bind in vivo primarily to the ends of plant chromosomes, which consist of long stretches of telomeric repeats.     

Expansion of protein domain repeats

Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.