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Principal Investigator: Jeanine Faye Amacher
Organization: WESTERN WASHINGTON UNIVERSITY
Fiscal Year: 2024
Award: $382,912
Funding agency: National Institute of General Medical Sciences
Project Summary. Sortase enzymes are cysteine transpeptidases located on the cell surface of Gram-positive
bacteria, which attach a variety of proteins to the peptidoglycan cell wall. The first sortase discovered, the Class
A sortase from Staphylococcus aureus (saSrtA), is widely used in sortase-mediated ligation (SML) or sortagging
protein engineering applications. This is a versatile protein with numerous human health and disease
implications; recent examples include the use of an engineered saSrtA to identify and disrupt amyloid-b
aggregates in a neurodegeneration model and to construct multi-arm SARS-CoV-2 neutralizing nanobodies.
SrtA is also a potential antibiotic target, as it is an essential protein in these pathogenic bacteria. However, recent
work from ourselves and others reveals that the stringent target selectivity of saSrtA is not shared amongst other
Class A sortases, e.g., those from the Streptococcus or Listeria genera. Furthermore, other classes of sortases
are extremely understudied with respect to SrtA proteins, and could provide useful tools for SML applications.
Class B sortases (SrtBs), for example, recognize a different motif sequence, which may be exploited to expand
the capabilities of SML. Finally, early evidence suggests that the transmembrane region of saSrtA may play an
important role in the catalytic efficiency of this enzyme, but this has not been thoroughly investigated.
The overall objectives in this application are to (i) dissect selectivity and activity determinants in sortase
enzymes from multiple classes, including the transmembrane regions of sortases and/or their substrates, and
(ii) use bacterial display to create a high throughput assay to measure sortase selectivity, which would be
applicable broadly to all classes of sortases. The central hypothesis is that we can use protein biochemistry,
chemical biology, and computational biology to better understand these enzymes. The rationale of this project is
that a deep understanding of the general characteristics of sortase biology can be leveraged to develop new
SML strategies for protein engineering, and assist in targeted antibiotic design to improve human health. The
central hypothesis will be tested by pursuing three specific aims: 1) dissect selectivity and activity determinants
in SrtA enzymes by characterizing differences in protein dynamics and elucidating extended motif preferences,
as well as develop a high throughout bacterial display assay for sortase selectivity, 2) advance a molecular
understanding of SrtB activities in order to improve reaction rates, and 3) characterize the role of the
transmembrane regions of sortase and its substrates in catalysis. All aims will utilize protein biochemistry,
structural biology, chemical biology, and computational biology. The research proposed in this application is
innovative in that we will be developing new assays and applying techniques not previously used with sortase
enzymes, continuing our work as a productive collaborative team to investigate aspects of sortase enzymes not
currently well understood. The proposed research is significant because our work will inform next-generation
SML methods and can be applied to the development of new antibiotics for Gram-positive bacterial pathogens.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV vaccine><Affect><Affinity><Africa South of the Sahara><Amines><Antibiotic Agents><Antibiotic Drugs><Antibiotic Resistance><Antibiotics><Antimicrobial Resistance><Assay><B anthracis><B. anthracis><Bacillus anthracis><Bacteria><Basic Research><Basic Science><Binding><Binding Proteins><Bioassay><Biochemical><Biological Assay><Biology><Biotin><C-terminal><COVID-19 vaccine><COVID-19 virus><COVID19 virus><Catalysis><Cell Wall><Cell surface><Cessation of life><Characteristics><Chemicals><Chimera><Chimera organism><Closure by Ligation><CoV-2><CoV2><Computational Biology><Cysteine><Death><Development><Diagnostic tests><Dimerization><Disease><Disorder><Drug resistance><Engineering><Enzyme Gene><Enzymes><Event><FRET><Fluorescence Resonance Energy Transfer><Foundations><Förster Resonance Energy Transfer><Genetic Alteration><Genetic Change><Genetic defect><Gram-Positive Bacteria><Half-Cystine><Hand><Health><High Throughput Assay><Human><In Vitro><Knowledge><L monocytogenes><L-Cysteine><L. monocytogenes><Length><Ligand Binding Protein><Ligand Binding Protein Gene><Ligation><Lipids><Listeria><Listeria monocytogenes><MRSA><Maps><Measures><Mediating><Methicillin Resistant S. Aureus><Methodology><Methods><Miscellaneous Antibiotic><Mission><Modeling><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Interaction><Molecular Stereochemistry><Murein><Mutation><NIGMS><National Institute of General Medical Sciences><Nerve Degeneration><Neuron Degeneration><Peptide Synthesis><Peptides><Peptidoglycan><Peptidyl Transferases><Peptidyl Translocases><Peptidyltransferase><Phase><Play><Position><Positioning Attribute><Productivity><Protein Binding><Protein Biochemistry><Protein Dimerization><Protein Dynamics><Protein Engineering><Protein Family><Protein/Amino Acid Biochemistry><Proteins><Public Health><Reaction><Reporting><Research><Resistance to antibiotics><Resistant S. Pneumoniae><Resistant Streptococcus pneumoniae><Resistant to antibiotics><Role><S aureus><S pyogenes><S. aureus><S. pyogenes><SARS corona virus 2><SARS-CO-V2><SARS-COVID-2><SARS-CoV-2><SARS-CoV-2 vaccine><SARS-CoV2><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-coronavirus-2 vaccine><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Series><Severe Acute Respiratory Coronavirus 2><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV 2 vaccine><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome coronavirus 2 vaccine><Severe acute respiratory syndrome related corona virus 2><Specificity><Staph aureus><Staphylococcus aureus><Strepavidin><Streptavidin><Streptococcus><Streptococcus Group A><Streptococcus pyogenes><Sub-Saharan Africa><Subsaharan Africa><Substrate Interaction><System><TM Domain><Techniques><Testing><Transmembrane Domain><Transmembrane Region><Transpeptidases><Variant><Variation><Vitamin H><Work><Wuhan coronavirus><abeta accumulation><abeta aggregation><amine><amyloid beta accumulation><amyloid beta aggregation><amyloid β accumulation><amyloid β aggregation><anthracis><anti-microbial resistant><antibiotic design><antibiotic drug resistance><antibiotic resistant><arm><aβ accumulation><aβ aggregation><bacteria pathogen><bacterial pathogen><bound protein><chemical reaction rate><chimeras><coenzyme R><computer biology><conformation><conformational><conformational state><conformationally><conformations><coronavirus disease 2019 vaccine><coronavirus disease 2019 virus><coronavirus disease-19 vaccine><coronavirus disease-19 virus><developmental><drug resistant><evaluate vaccines><flexibility><flexible><genetic protein engineering><genome mutation><hCoV19><hands><high throughput screening><improved><innovate><innovation><innovative><methicillin resistance Staphylococcus aureus><methicillin resistant Staphylococcus aureus><methicillin resistant strains of Staphylococcus aureus><molecular dynamics><nCoV vaccine><nCoV-19 vaccine><nCoV19 vaccine><nCoV2><nanobodies><nanobody><nanodisk><neural degeneration><neurodegeneration><neurodegenerative><neurological degeneration><neuronal degeneration><next generation><pathogen><pathogenic bacteria><preference><protein design><protein purification><reaction rate><resistance to Drug><resistance to anti-microbial><resistant strains of S. Pneumoniae><resistant to Drug><resistant to antimicrobial><sdAb><single domain antibodies><social role><sortase><srtA gene product><structural biology><therapeutic target><tool><transacylation><vaccine against 2019-nCov><vaccine against COVID-19><vaccine against SARS-CoV-2><vaccine against SARS-coronavirus-2><vaccine against Severe Acute Respiratory Syndrome CoV 2><vaccine against Severe acute respiratory syndrome coronavirus 2><vaccine candidates against SARS-CoV-2><vaccine evaluation><vaccine for novel coronavirus><vaccine screening><vaccine testing><vaccines preventing COVID><vaccines to prevent COVID>