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Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.

We report here the crystal structure of the minimal ligand‐binding segment of the Staphylococcus aureus MSCRAMM, clumping factor A. This fibrinogen‐binding segment contains two similarly folded domains. The fold observed is a new variant of the immunoglobulin motif that we have called DE‐variant or the DEv‐IgG fold. This subgroup includes the ligand‐binding domain of the collagen‐binding S.aureus MSCRAMM CNA, and many other structures previously classified as jelly rolls. Structure predictions suggest that the four fibrinogen‐binding S.aureus MSCRAMMs identified so far would also contain the same DEv‐IgG fold. A systematic docking search using the C‐terminal region of the fibrinogen γ‐chain as a probe suggested that a hydrophobic pocket formed between the two DEv‐IgG domains of the clumping factor as the ligand‐binding site. Mutagenic substitution of residues Tyr256, Pro336, Tyr338 and Lys389 in the clumping factor, which are proposed to contact the terminal residues 408 AGDV 411 of the γ‐chain, resulted in proteins with no or markedly reduced affinity for fibrinogen.

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes ( pgfM1 , pgfE and pgfM2 ) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized Δ pgfS strain, inactivation of pgfM1 , pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S . mutans .

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A₃VP₁ fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A₃ repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P₁ repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A₃ and P₁ helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Hereditary cancer syndromes account for approximately 5%–10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N‐terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in‐cell fluorescent‐based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign. We identified a variant within the PMS2 N‐terminal domain, p.Asn335Ser, that resulted in a protein that lacked ATPase activity, yet appears to be proficient in MMR. To understand this functional dichotomy, we performed biophysical and structural studies and noted that the p.Asn335Ser variant enzyme binds ATP but does not hydrolyze the substrate to ADP.

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the [alpha]- and [beta]-crystallins. Here, we investigated the effects of the deamidation of [alpha]A- and [alpha]B-crystallins on their interaction with [beta]A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT [alpha]A- and WT [alpha]B-crystallins and their deamidated mutants with [beta]A3-crystallin. The deamidated mutants of [alpha]A-crystallin ([alpha]A N101D and [alpha]A N123D) displayed lower adherence propensity for [beta]A3-crystallin relative to the binding affinity shown by WT [alpha]A-crystallin. Among [alpha]B-crystallin mutants, [alpha]B N78D displayed higher adherence propensity whereas [alpha]B N146D mutant showed slightly lower binding affinity for [beta]A3-crystallin relative to that shown by WT [alpha]B-crystallin. Under the in vivo condition (FLIM-FRET), both [alpha]A-deamidated mutants ([alpha]A N101D and [alpha]A N123D) exhibited strong interaction with [beta]A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT [alpha]A-crystallin (18±4%). Similarly, the [alpha]B N78D and [alpha]B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with [beta]A3-crystallin compared to 18±4% FRET efficiency of WT [alpha]B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of [alpha]A- and [alpha]B-crystallins with [beta]A3-crystallin suggested that interaction sites most likely reside in the [alpha]A CTE and [alpha]B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT [alpha]A- and WT[alpha]B-crystallins, the deamidated mutants showed strong interactionfor [beta]A3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of [alpha]-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 Å) crystal structure of the A₃VP₁ fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 Å) through the interaction of two noncontiguous regions in the primary sequence. The A₃ repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P₁ repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A₃ and P₁ helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 A) crystal structure of the A...VP... fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 A) through the interaction of two noncontiguous regions in the primary sequence. The A... repeat of the alanine-rich domain adopts an extended α-helix that intertwines with the P... repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A... and P... helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length. (ProQuest: ... denotes formulae/symbols omitted.)