Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.     

Mutational analysis of the KIX domain of CBP reveals residues critical for SREBP binding

Structure-based mutagenesis was used to probe the binding surface for the activation domain of sterol-responsive element binding protein (SREBP) in the KIX domain of CREB binding protein. A set of conserved residues scattering in the alpha2 helix and the extended C-terminal region of alpha 3 helix in the KIX domain including two arginines previously characterized as a hot spot for cofactor-mediated methylation was shown to be crucial for SREBP-KIX interaction, and was not essential for phosphorylated KID recognition. Therefore, our results suggest the existence of a SREBP binding site formed by positively charged residues in the C-terminal part of the extended alpha 3 helix of the KIX domain distinct from the previously identified phosphorylated KID binding site.     

Mutational analysis of human NOD1 and NOD2 NACHT domains reveals different modes of activation

Nucleotide-binding oligomerization domain-containing protein (NOD)1 and NOD2 are intracellular pattern recognition receptors (PRRs) of the nucleotide-binding domain and leucine-rich repeat containing (NLR) gene family involved in innate immune responses. Their centrally located NACHT domain displays ATPase activity and is necessary for activation and oligomerization leading to inflammatory signaling responses. Mutations affecting key residues of the ATPase domain of NOD2 are linked to severe auto-inflammatory diseases, such as Blau syndrome and early-onset sarcoidosis. By mutational dissection of the ATPase domain function, we show that the NLR-specific extended Walker B box (DGhDE) can functionally replace the canonical Walker B sequence (DDhWD) found in other ATPases. A requirement for an intact Walker A box and the magnesium-co-ordinating aspartate of the classical Walker B box suggest that an initial ATP hydrolysis step is necessary for activation of both NOD1 and NOD2. In contrast, a Blau-syndrome associated mutation located in the extended Walker B box of NOD2 that results in higher autoactivation and ligand-induced signaling does not affect NOD1 function. Moreover, mutation of a conserved histidine in the NACHT domain also has contrasting effects on NOD1 and NOD2 mediated NF-κB activation. We conclude that these two NLRs employ different modes of activation and propose distinct models for activation of NOD1 and NOD2.     

Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation

In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.     

Mutational analysis of plant cap-binding protein eIF4E reveals key amino acids involved in biochemical functions and potyvirus infection

The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1¹ and mo1² against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1¹ or mo1² varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.     

Mutational analysis of chicken interleukin 2

Chicken interleukin 2 (chIL-2) has low, but significant, homology to both mammalian IL-2 and mammalian IL-15. In view of its unique phylogenetic position and potential use as a vaccine adjuvant, a detailed mutational analysis for critical functional sites was undertaken. It was found that Asp17 is a critical N terminal contact site for binding to the putative chIL-2 receptor, which is similar to results obtained for mammalian IL-2 and IL-15. Analysis of the C terminus did not reveal a single critical amino acid. However, deletion mutant studies demonstrated that removal of C terminal amino acids yielded proteins with decreased bioactivity and that this decrease was a function of the number and kind of amino acids removed. This study is the first non-mammalian IL-2 mutational analysis and proposes a model for the interaction between chIL-2 and its receptor.     

Mutational analysis of the antizyme-binding element reveals critical residues for the function of ornithine decarboxylase

Background

Ornithine decarboxylase (ODC), the key enzyme in the polyamine biosynthetic pathway, is highly regulated by antizymes (AZs), small proteins that bind and inhibit ODC and increase its proteasomal degradation. Early studies delimited the putative AZ-binding element (AZBE) to the region 117-140 of ODC. The aim of the present work was to study the importance of certain residues of the region 110-142 that includes the AZBE region for the interaction between ODC and AZ1 and the ODC functionality.

Methods

Computational analysis of the protein sequences of the extended AZBE site of ODC and ODC paralogues from different eukaryotes was used to search for conserved residues. The influence of these residues on ODC functionality was studied by site directed mutagenesis, followed by different biochemical techniques.

Results

The results revealed that: a) there are five conserved residues in ODC and its paralogues: K115, A123, E138, L139 and K141; b) among these, L139 is the most critical one for the interaction with AZs, since its substitution decreases the affinity of the mutant protein towards AZs; c) all these conserved residues, with the exception of A123, are critical for ODC activity; d) substitutions of K115, E138 or L139 diminish the formation of ODC homodimers.

Conclusions

These results reveal that four of the invariant residues of the AZBE region are strongly related to ODC functionality.

General significance

This work helps to understand the interaction between ODC and AZ1, and describes various new residues involved in ODC activity, a key enzyme for cell growth and proliferation.     

Mutational analysis of the ras converting enzyme reveals a requirement for glutamate and histidine residues

The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.     

Mutational analysis of SARS-CoV-2 variants of concern reveals key tradeoffs between receptor affinity and antibody escape

SARS-CoV-2 variants with enhanced transmissibility represent a serious threat to global health. Here we report machine learning models that can predict the impact of receptor-binding domain (RBD) mutations on receptor (ACE2) affinity, which is linked to infectivity, and escape from human serum antibodies, which is linked to viral neutralization. Importantly, the models predict many of the known impacts of RBD mutations in current and former Variants of Concern on receptor affinity and antibody escape as well as novel sets of mutations that strongly modulate both properties. Moreover, these models reveal key opposing impacts of RBD mutations on transmissibility, as many sets of RBD mutations predicted to increase antibody escape are also predicted to reduce receptor affinity and vice versa. These models, when used in concert, capture the complex impacts of SARS-CoV-2 mutations on properties linked to transmissibility and are expected to improve the development of next-generation vaccines and biotherapeutics.     

Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer

The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.     

Mutational analysis of human papillomavirus type 16 E7 functions.

The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.     

Mutational activation of ErbB2 reveals a new protein kinase autoinhibition mechanism

Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.     

Mutational analysis reveals a dual role of Mdm2 acidic domain in the regulation of p53 stability

The exact role of the central acidic domain of Mdm2 in p53 degradation remains unclear. We therefore performed a systematic and comprehensive analysis of the acidic domain using a series of short deletions and found that only a minor part of the domain was indispensable for Mdm2-mediated p53 ubiquitylation. Moreover, we identified a short stretch of acidic amino acids required for p53 degradation but not ubiquitylation, indicating that, in addition to p53 ubiquitylation, the acidic domain might be involved in a critical post-ubiquitylation step in p53 degradation. Rather than representing a single functional domain, different parts of the acidic region perform separate functions in p53 degradation, suggesting that it might be possible to therapeutically target them independently.     

Mutational analysis of G-protein coupled receptor--FFA2

As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.     

Mutational analysis reveals distinct features of the Nox4-p22 phox complex

The integral membrane protein p22(phox) forms a heterodimeric enzyme complex with NADPH oxidases (Noxs) and is required for their catalytic activity. Nox4, a Nox linked to cardiovascular disease, angiogenesis, and insulin signaling, is unique in its ability to produce hydrogen peroxide constitutively. To date, p22(phox) constitutes the only identified regulatory component for Nox4 function. To delineate structural elements in p22(phox) essential for formation and localization of the Nox4-p22(phox) complex and its enzymatic function, truncation and point mutagenesis was used. Human lung carcinoma cells served as a heterologous expression system, since this cell type is p22(phox)-deficient and promotes cell surface expression of the Nox4-p22(phox) heterodimer. Expression of p22(phox) truncation mutants indicates that the dual tryptophan motif contained in the N-terminal amino acids 6-11 is essential, whereas the C terminus (amino acids 130-195) is dispensable for Nox4 activity. Introduction of charged residues in domains predicted to be extracellular by topology modeling was mostly tolerated, whereas the exchange of amino acids in predicted membrane-spanning domains caused loss of function or showed distinct differences in p22(phox) interaction with various Noxs. For example, the substitution of tyrosine 121 with histidine in p22(phox), which abolished Nox2 and Nox3 function in vivo, preserved Nox4 activity when expressed in lung cancer cells. Many of the examined p22(phox) mutations inhibiting Nox1 to -3 maturation did not alter Nox4-p22(phox) association, further accenting the differences between Noxs. These studies highlight the distinct interaction of the key regulatory p22(phox) subunit with Nox4, a feature which could provide the basis for selective inhibitor development.     

Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF

Bacterial condensin MukBEF is essential for global folding of the Escherichia coli chromosome. MukB, a SMC (structural maintenance of chromosome) protein, comprises the core of this complex and is responsible for its ATP‐modulated DNA binding and reshaping activities. MukF serves as a kleisin that modulates MukB–DNA interactions and links MukBs into macromolecular assemblies. Little is known about the function of MukE. Using random mutagenesis, we generated six loss‐of‐function point mutations in MukE. The surface mutations clustered in two places. One of them was at or close to the interface with MukF while the other was away from the known interactions of the protein. All loss‐of‐function mutations affected focal localization of MukBEF in live cells. In vitro, however, only some of them interfered with the assembly of MukBEF into a complex or the ability of MukEF to disrupt MukB–DNA interactions. Moreover, some MukE mutants were able to join intracellular foci formed by endogenous MukBEF and most of the mutants were efficiently incorporated into MukBEF even in the presence of endogenous MukE. These data reveal that focal localization of MukBEF involves other activities besides DNA binding and that MukE plays a central role in them.     

Mutational analysis of ErbB2 intracellular localization

In the present study, the sub-cellular localization of ErbB2 and its mutants expressed as GFP-tagged proteins in MCF-7 cells or endogenous ErbB2 in SKBR3 cells was examined. The data presented here demonstrate that the full-length ErbB2 was localized at the cytoplasmic membrane and ErbB2 ICD localized in the nucleus predominantly. The sequence of ErbB2 ICD contains the information supporting its nuclear translocation and cytoplasmic retention. A region (residues 721–970) harboring an arginine triplet is essential for the cytoplasmic trafficking of ErbB2. The results indicate that differential sub-cellular localization of ErbB2 ICD and the full-length ErbB2 is dependent on their structural determinants. The present results give initial clues for further analysis of the mechanism of ErbB2 intracellular localization.