Chemical Modification Studies of Gelonin - Involvement of Arginine Residues in Biological-Activity

Gelonin inhibits protein synthesis by inactivating the eukaryotic 60 S ribosomal subunit by an unknown mechanism. The protein was purified in high yield by a new method using Cibacron blue F3GA-Sepharose. Chemical modification studies reveal that arginine residues are essential for biological activity.

Interaction Between Two Residues in the Inter-Domain Interface of Escherichia coli Peptidase N Modulates Catalytic Activity

The role of interaction between Asn259 (catalytic domain) with Gln821 (C-terminal domain) in PeptidaseN was investigated. The k(cat) of PeptidaseN containing Asn259Asp or Gln821Glu is enhanced whereas it is suppressed in Asn259AspGln821Glu. Structural analysis shows this interaction to change the relative disposition of active site residues, which modulates catalytic activity.

Chemical modification studies on Abrus agglutinin Involvement of tryptophan residues in sugar binding

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydratebinding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2- hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.

Stereochemical restrictions on the occurrence of amino acid residues in the collagen structure

The primary structure of collagen is characterized by the repeating tripeptide sequence (Gly-R2-R3)n. The results of theoretical studies, carried out using contact criteria to compute the stereochemically allowed orientations for various side chains at locations 2 and 3, are reported here. It is found that side chains with only γ-atoms, as in valine, serine and threonine, or with only one δ-methyl group, as in isoleucine, can occur equally well at locations 2 and 3, as is actually the case in collagen. Side chains with two Cδ-atoms, as in leucine and phenyl-alanine, can also be accommodated at both positions. However, if they occur as R3 their freedom of orientation is severely restricted in the presence of a proline residue as R2 in a neighbouring chain. If water molecules bound to the chains of the triple helix are assumed to be present, then location 3 is virtually impossible for leucine and phenylalanine residues. Location 2 is, however, unaffected, and their presence as R2 can help to shield the water molecules from disturbance by the solvent medium. This may be the reason for the preferential occurrence of Leu and Phe residues in location 2 in the collagen triplets, although the polypeptides (Gly-Pro-Leu)n and (Gly-Pro-Phe)n form collagen-like structures.

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed, H134N and H147N SHMTs had K-m values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the k(cat) values were markedly decreased, H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme, Increasing concentrations of PLP (up to 500 mu M) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions, H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding, Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface, H150N SHMT was obtained in a tetrameric form with bound PLP, However, the mutant had very little enzyme activity (<2%). The k(cat)/K-m values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and SS-fold less compared with wild type enzyme, Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H-4-folate. However, it could form an external aldimine with serine and glycine, The wild type and the mutant enzyme had similar K-d values for serine and glycine, These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Recognition of Interaction Interface Residues in Low-Resolution Structures of Protein Assemblies Solely from the Positions of C alpha Atoms

Background: The number of available structures of large multi-protein assemblies is quite small. Such structures provide phenomenal insights on the organization, mechanism of formation and functional properties of the assembly. Hence detailed analysis of such structures is highly rewarding. However, the common problem in such analyses is the low resolution of these structures. In the recent times a number of attempts that combine low resolution cryo-EM data with higher resolution structures determined using X-ray analysis or NMR or generated using comparative modeling have been reported. Even in such attempts the best result one arrives at is the very course idea about the assembly structure in terms of trace of the C alpha atoms which are modeled with modest accuracy. Methodology/Principal Findings: In this paper first we present an objective approach to identify potentially solvent exposed and buried residues solely from the position of C alpha atoms and amino acid sequence using residue type-dependent thresholds for accessible surface areas of C alpha. We extend the method further to recognize potential protein-protein interface residues. Conclusion/Significance: Our approach to identify buried and exposed residues solely from the positions of C alpha atoms resulted in an accuracy of 84%, sensitivity of 83-89% and specificity of 67-94% while recognition of interfacial residues corresponded to an accuracy of 94%, sensitivity of 70-96% and specificity of 58-94%. Interestingly, detailed analysis of cases of mismatch between recognition of interface residues from C alpha positions and all-atom models suggested that, recognition of interfacial residues using C alpha atoms only correspond better with intuitive notion of what is an interfacial residue. Our method should be useful in the objective analysis of structures of protein assemblies when positions of only C alpha positions are available as, for example, in the cases of integration of cryo-EM data and high resolution structures of the components of the assembly.

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Role of histidine residues in EcoP15I DNA methyltransferase activity as probed by chemical modification and site-directed mutagenesis

Towards understanding the catalytic mechanism of M.EcoP15I [EcoP15I MTase (DNA methyltransferase); an adenine methyltransferase], we investigated the role of histidine residues in catalysis. M.EcoP15I, when incubated with DEPC (diethyl pyrocarbonate), a histidine-specific reagent, shows a time- and concentration-dependent inactivation of methylation of DNA containing its recognition sequence of 5'-CAGCAG-3'. The loss of enzyme activity was accompanied by an increase in absorbance at 240 nm. A difference spectrum of modified versus native enzyme shows the formation of N-carbethoxyhistidine that is diminished by hydroxylamine. This, along with other experiments, strongly suggests that the inactivation of the enzyme by DEPC was specific for histidine residues. Substrate protection experiments show that pre-incubating the methylase with DNA was able to protect the enzyme from DEPC inactivation. Site-directed mutagenesis experiments in which the 15 histidine residues in the enzyme were replaced individually with alanine corroborated the chemical modification studies and established the importance of His-335 in the methylase activity. No gross structural differences were detected between the native and H335A mutant MTases, as evident from CD spectra, native PAGE pattern or on gel filtration chromatography. Replacement of histidine with alanine residue at position 335 results in a mutant enzyme that is catalytically inactive and binds to DNA more tightly than the wild-type enzyme. Thus we have shown in the present study, through a combination of chemical modification and site-directed mutagenesis experiments, that His-335 plays an essential role in DNA methylation catalysed by M.EcoP15I.

Conformational characteristics of asparaginyl residues in proteins

Backbone conformations at 1064 asparaginyl residues in 123 non-homologous, high-resolution X-ray structures of proteins were analysed. Asn adopts conformations in left-handed x-helical region and other partially allowed regions in the Ramachandran map more readily than any other non-glycyl residue. Asn conformational clusters in the (phi,psi) regions of left-handed alpha-helix, right-handed alpha-helix and extended (beta) strands were investigated in detail for their occurrence in various secondary structures, especially in beta-turn regions. Preferences were observed for Asn conformations in different positions in various beta-turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II beta-turns. Asn conformations at the N-cap positions of helices strongly prefer extended conformation than alpha(L), which seems to be characteristic of non-glycyl residues at that position. In the linkers connecting two extended strands and those connecting an alpha-helix and an extended strand, Asn with alpha(L) or alpha(R) conformation is more favoured than Asn with the beta-conformation. Analysis of Asn-Asn doublets and Asn-X-Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn-rich regions in proteins such as malaria parasite coat protein. (C) Munksgaard 1994.

Disallowed Ramachandran Conformations of Amino Acid Residues in Protein Structures

An analysis of the nature and distribution of disallowed Ramachandran conformations of amino acid residues observed in high resolution protein crystal structures has been carried out. A data set consisting of 110 high resolution, non-homologous, protein crystal structures from the Brookhaven Protein Data Bank was examined. The data set consisted of a total of 18,708 non-Gly residues, which were characterized on the basis of their backbone dihedral angles (φ, ψ). Residues falling outside the defined “broad allowed limits” on the Ramachandran map were chosen and the reportedB-factor value of the α-carbon atom was used to further select well defined disallowed conformations. The conformations of the selected 66 disallowed residues clustered in distinct regions of the Ramachandran map indicating that specific φ, ψ angle distortions are preferred under compulsions imposed by local constraints. The distribution of various amino acid residues in the disallowed residue data set showed a predominance of small polar/charged residues, with bulky hydrophobic residues being infrequent. As a further check, for all the 66 cases non-hydrogen van der Waals short contacts in the protein structures were evaluated and compared with the ideal “Ala-dipeptide” constructed using disallowed dihedral angle (φ, ψ) values. The analysis reveals that short contacts are eliminated in most cases by local distortions of bond angles. An analysis of the conformation of the identified disallowed residues in related protein structures reveals instances of conservation of unusual stereochemistry.

Identification of Specific DNA Binding Residues in the TCP Family of Transcription Factors in Arabidopsis

The TCP transcription factors control multiple developmental traits in diverse plant species. Members of this family share an similar to 60-residue-long TCP domain that binds to DNA. The TCP domain is predicted to form a basic helix-loop-helix ( bHLH) structure but shares little sequence similarity with canonical bHLH domain. This classifies the TCP domain as a novel class of DNA binding domain specific to the plant kingdom. Little is known about how the TCP domain interacts with its target DNA. We report biochemical characterization and DNA binding properties of a TCP member in Arabidopsis thaliana, TCP4. We have shown that the 58-residue domain of TCP4 is essential and sufficient for binding to DNA and possesses DNA binding parameters comparable to canonical bHLH proteins. Using a yeast-based random mutagenesis screen and site-directed mutants, we identified the residues important for DNA binding and dimer formation. Mutants defective in binding and dimerization failed to rescue the phenotype of an Arabidopsis line lacking the endogenous TCP4 activity. By combining structure prediction, functional characterization of the mutants, and molecular modeling, we suggest a possible DNA binding mechanism for this class of transcription factors.

A structural basis for the role of nucleotide specifying residues in regulating the Rv1625c adenylyl cyclase oligomerization of the from M-tuberculosis

The Rv1625c Class III adenylyl cyclase from Mycobacterium tuberculosis is a homodimeric enzyme with two catalytic centers at the dimer interface, and shows sequence similarity with the mammalian adenylyl and guanylyl cyclases. Mutation of the substrate-specifying residues in the catalytic domain of Rv1625c, either independently or together, to those present in guanylyl cyclases not only failed to confer guanylyl cyclase activity to the protein, but also severely abrogated the adenylyl cyclase activity of the enzyme. Biochemical analysis revealed alterations in the behavior of the mutants on ion-exchange chromatography, indicating differences in the surface-exposed charge upon mutation of substrate-specifying residues. The mutant proteins showed alterations in oligomeric status as compared to the wild-type enzyme, and differing abilities to heterodimerize with the wild-type protein. The crystal structure of a mutant has been solved to a resolution of 2.7 angstrom. On the basis of the structure, and additional biochemical studies, we provide possible reasons for the altered properties of the mutant proteins, as well as highlight unique structural features of the Rv1625c adenylyl cyclase. (c) 2005 Elsevier Ltd. All rights reserved.