Phosphosite conservation in single domain orthologs versus paralogs: a way to combine differential regulation with redundant core functions

Evolutionary conservation for structure function relations is commonly accepted. Here we hypothesize that closely related single domain paralogous proteins, having similar expression profiles and redundant biochemical core functions, additionally evolved to allow and maintain isoform specific differential regulation by single conserved amino acid substitutions. To substantiate this, we considered two families of closely related actin binding proteins combined with data mining of phosphorylated residues in human and mouse proteins. We show that such residues are identical in other orthologs whereas paralogs have a different, but also conserved, non-phosphorylatable residue at the equivalent positions.     

Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17-24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.     

Profiling of Substrate Specificity of SARS-CoV 3CLpro

Background

The 3C-like protease (3CL^pro) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection.

Methodology/Principal Findings

To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3'' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1'' position prefers small residues, while P2'' and P3'' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3'' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence.

Conclusions/Significance

Our results demonstrated a strong structure-activity relationship between the 3CL^pro and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.     

Correlation of structure with transforming activity of the P21 proteins with substitutions at positions 12 and 16

The structural effects of amino acid substitutions at positions 12 and 16 in the amino-terminal segment (Tyr 4-Ala 18) of the ras-oncogene-encoded P21 proteins have been investigated using conformational energy analysis. The P21 protein with Val at position 12 and Lys at position 16 is known to have high transforming ability, while the P21 protein with Val at position 12 and Asn at position 16 is known to have poor transforming ability, similar to that of the normal protein (with Gly at 12 and Lys at 16.) The current results demonstrate a significant conformational change at position 15 induced by the substitution of Asn for Lys at position 16, which could explain this alteration in transformation potential. These findings are consistent with previous results suggesting the existence of a normal and a malignancy-causing conformation for the P21 proteins and suggest that the critical transforming region may encompass residues 12–15.     

Cuttlefish spermatid-specific protein T. Molecular characterization of two variants T1 and T2, putative precursors of sperm protamine variants Sp1 and Sp2

In cuttlefish, as in selachians and mammals, spermiogenesis is characterized by the double nuclear protein transition histones----intermediate protein (protein T)----protamine (protein Sp). The cuttlefish protein T, which consists of two structural variants phosphorylated at different degrees, is the first invertebrate spermatid-specific protein to be fully characterized and sequenced. The primary structures of these two variants were established from sequence analysis and mass spectrometric data of the proteins and their fragments. T1 and T2 are two highly related proteins of 78 and 77 residues, respectively, which differ only by four conservative substitutions, two inversions Ser in equilibrium with Arg, and the deletion of 1 residue of arginine in variant T2. The asymmetrical distribution of the hydrophobic and basic residues determines two well defined domains: an amino-terminal domain (residues 1-21) devoid of arginine and aromatic residues and containing all the aliphatic hydrophobic residues and a highly basic carboxyl-terminal domain (residues 22-77 or 78) that contains 77% of arginine, all the tyrosine residues, and most of the phosphorylated serine residues present in the protein. The complete structural identity of the basic carboxyl-terminal domain of spermatidal proteins T1 and T2 with the protamine variants Sp1 and Sp2 isolated from cuttlefish spermatozoa strongly suggests that T1 and T2 could be precursors of Sp1 and Sp2, respectively.     

The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.     

The recognition component of the N-end rule pathway.

The N-end rule-based degradation signal, which targets a protein for ubiquitin-dependent proteolysis, comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We report the isolation and functional analysis of a gene (UBR1) for the N-end recognizing protein of the yeast Saccharomyces cerevisiae. UBR1 encodes a approximately 225 kd protein with no significant sequence similarities to other known proteins. Null ubr1 mutants are viable but are unable to degrade the substrates of the N-end rule pathway. These mutants are partially defective in sporulation and grow slightly more slowly than their wild-type counterparts. The UBR1 protein specifically binds in vitro to proteins bearing amino-terminal residues that are destabilizing according to the N-end rule, but does not bind to otherwise identical proteins bearing stabilizing amino-terminal residues.     

The amino acid sequence of the Tetrahymena calmodulin which specifically interacts with guanylate cyclase

The amino acid sequence of the Tetrahymena calmodulin was determined. The protein is composed of 147 amino acids and the amino-terminal is acetylated. Compared to bovine brain calmodulin, there were eleven substitutions and one deletion of amino acid residues. The substitutions and deletion were concentrated in the carboxyl-terminal half of the molecule. Among the substitutions, those at positions 86 (Arg → Ile), 135 (Gln → His) and 143 (Gln → Arg) may introduce the functional difference. The deletion occurred near the carboxyl-terminal, this region being assumed to be exposed to the surface area (R.H. Kretsinger and C.D. Barry (1975)). The change in the sequence at this terminal region may be attributable to the specific activation of guanylate cyclase.     

The primary structure of skeletal muscle myosin heavy chain: I. Sequence of the amino-terminal 23 kDa fragment

Subfragment-1 was prepared from adult chicken pectoralis myosin by limited digestion with alpha-chymotrypsin, and an amino-terminal 23 kDa fragment of the heavy chain was obtained by digesting the subfragment-1 with trypsin. The 205-residue sequence of the fragment was determined by sequencing its cyanogen bromide, tryptic, and chymotryptic peptides. The amino-terminal alpha-amino group of the fragment was acetylated, and two methylated lysines; epsilon-N-monomethyllysine and epsilon-N-trimethyllysine were recognized at the 35th and 130th positions, respectively, as in rabbit skeletal myosin. Comparing the 205-residue sequence of the skeletal myosin with those of cardiac, and gizzard myosins from chicken, considerable differences are recognized, especially in the amino-terminal region, but strong homologies are observed around the reactive lysine residue, around the epsilon-N-trimethyllysine residue, and around the consensus sequence of GXXGXGKT for nucleotide-binding proteins. On the other hand, only 12 amino acid substitutions are recognized between adult and embryonic skeletal myosins, allowing for the post-translational methylation.     

Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization

Assembly of monomeric amyloid beta-protein (A beta) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A beta 40 and A beta 42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A beta 40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A beta 42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A beta alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A beta 42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. A beta 40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. A beta 42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control A beta oligomerization and show that these controlling elements differ between A beta 40 and A beta 42.     

Mutational analysis of human hydroxysteroid sulfotransferase SULT2B1 isoforms reveals that exon 1B of the SULT2B1 gene produces cholesterol sulfotransferase,whereas exon 1A yields pregnenolone sulfotransferase

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.     

Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning

Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the food-borne pathogen Listeria monocytogenes. To elucidate which residue(s) is responsible for this function, the antimicrobial activities of pediocin PA-1 mutants were evaluated and compared. Each of the 44 native codons was replaced with the NNK triplet oligonucleotide in a technique termed NNK scanning, and 35 mutations at each position were examined for antimicrobial activities using a modified colony overlay screening method. As a consequence, the functional responsibility of each residue was estimated by counting the number of active mutants, allowing us to identify candidate essential/variable residues. Activity was abrogated by many of the mutations at residues Y2, G6, C9, C14, C24, W33, G37, and C44, indicating that these residues may be essential. In contrast, activity was retained by almost all versions harboring mutations at K1, T8, G10, S13, G19, N28, and N41, indicating that these are functionally redundant residues. Sequence analysis revealed that only the wild type was active and 14 and 11 substitutions were inactive at G6 and C14, respectively, while 12 and 11 substitutions were active and 2 and 0 substitutions were inactive at T8 and K1, respectively. These findings suggest that NNK scanning is effective for determining essential and variable residues in pediocin PA-1, leading to an elucidation of structure-function relationships and to improvements in the antimicrobial function efficiently by peptide engineering.     

Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi,an Extremely Halophilic Microbe from Antarctica

The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.     

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.     

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.     

Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S

Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and localized the thrombin cleavage sites. Protein S contains 11 gamma-carboxyglutamic acid residues in the amino-terminal region (residues 1-36). This part of protein S is highly homologous to the corresponding parts in the other vitamin K-dependent clotting factors, whereas the region between residues 45 and 75 is not at all homologous to the other clotting factors. Thrombin cleaves two peptide bonds in this part of protein S, first at arginine 70 and then at arginine 52. The peptide containing residues 53-70 is released from protein S after thrombin cleavage. The amino-terminal fragment, residues 1-52, is linked to the large carboxyl-terminal fragment by a disulfide bond, which involves cysteine 47. After residue 78, protein S is again homologous to factors IX and X and to proteins C and Z, but not to prothrombin. Position 95 is occupied by a beta-hydroxyaspartic acid residue.     

The amino acid sequence of tauropine dehydrogenase from the polychaete Arabella iricolor

The amino acid sequence of tauropine dehydrogenase (EC 1.5.1.23) from the polychaete Arabella iricolor was determined by automated sequencing of fragments obtained by cleavage with lysyl endopeptidase, endoproteinase Glu-C, and cyanogen bromide. The purified enzyme contained two isoforms that differ only in the 41st amino acid residue (Thr or Ile). Although the sequence contained eight Cys residues, intrachain disulfide bonds were not found. Two possible N-linked glycosylation sites occur in the sequences, but the enzyme does not appear to contain bound carbohydrates. Based on these data, the two isoforms of Arabella tauropine dehydrogenase are simple proteins consisted of 396 amino acid residues with calculated molecular masses of 43,085.7 Da (Thr41 isoform) and 43,097.8 Da (Ile41 isoform).