CRISPR-associated Proteins of the Apern Subtype and their Function in

CRISPR-associated Proteins of the
Apern Subtype and their Function in
a Newly Discovered Prokaryotic
Adaptive Immune System
Donald Alsbury1,2, Nathanael Lintner1,2,3,
Mark Young2,4, and Martin Lawrence1,2
of Chemistry and Biochemistry, 2Thermal Biology Institute,
3Molecular Biosciences Program, and 4Dept. of Plant Sciences and Plant Pathology,
Montana State University, Bozeman, MT
CRISPR-cas System
• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)
and associated genes are responsible for
bacterial and archaeal resistance to
specific phages
• CRISPRs are genomic elements present in
about half of sequenced bacterial genomes
and almost all of archaea
* denotes phage derived spacer sequences
• Spacers are derived from phage DNA
are necessary and responsible for observed viral resistance
• CAS genes code for proteins necessary for processing of RNA and degradation of
foreign DNA
• The majority of mechanistic studies take place on bacterial organisms, little is
known about the function of the archaeal domain
Sorek et al., Nature Reviews. Microbiology 6:181
Proposed Mechanism
• Polymerase transcribes CRISPR sequence
• RNA now termed pre-CRISPR RNA oftentimes assumes a hairpin secondary structure
due to rich guanine and cytosine regions
• CASCADE recognizes RNA and processes pre-crRNA
removing sections of the repeats leaving full spacer sequence and two halves of opposite
adjacent repeats
• CASCADE binds to CRISPR RNA serving as a guide to base pair to viral DNA
eventually leading to its degradation avoiding cell infection
A: Denaturing PAGE gel indicates sRNA
stays bound to CASCADE complex
Sorek et al., Nature Reviews. Microbiology 6:181
Brouns et al., Science 321:960
CRISPR Associated Complex for Antiviral Defense
• CASCADE is comprised of cas
proteins A-E in Escherichia coli
A: Representation of CRISPR loci in Escherichia coli
• Cas E is responsible for nuclease
activity initially processing the
• Processes and utilizes crRNA
for identification and degradation
of foreign phage DNA
Brouns et al., Science 321:960
B: SDS-PAGE of affinity purified protein
CRISPR-cas Subtypes
• Eight different CRISPR-cas subtypes:
determined by different repeat patterns, sets of
genes, and species ranges
• 45 distinct families of CRISPR-associated
proteins found in 200 prokaryotic genomes
• Proteins of different families can coexist within
diverse subtypes
• Cas 1 is the only protein found in every
CRISPR-cas system
• Systems undergo constant evolution
Haft et al., PLoS Computational Biology.
Apern Subtype
• Family of CRISPRs only found in archaea
• Named after Aeropyrum pernix in which it
is the only CRISPR-cas subtype present
• Found exclusively in achaea and the only
subtype present in chrenarchaea
• Genotypical difference: Repeats tend to be
a bit longer than other subtypes at around
66 base pairs
• Given abbreviation CSA
Jansen et al., Mol Microbial 6:1565
Sulfolobus solfataricus
• Thermophilic, acidophilic archaeon
• Optimal growth occurring at pH 2-3 and
temperatures between 75-80° C
• Inhabit Yellowstone hot pots
• Contain Apern CRISPR-cas
immune system
• Contains Cas 1-6
Csa 1-3, 5
Brumfield et al., Journal of Virology 12:5964
Cas proteins from Sulfolobus
solfataricus currently under study
• Csa1 sso-1391
• Csa2 sso-1442
• Csa3 sso-1445
• Cas5 sso-1441
S. solfataricus P2
3.0 Mbp
Csa 2
Crystallization and Structure Determination
Expressed in BL21(DE3)pLysS cells in ZYP-5025 (Studier)
Crystallized (P212121 4 chains per assymetric unit, 50% solvent content)
using hanging drop vapor diffusion, 1.9-2.1 M Na Formate, 0.1 M Na
Acetate pH 4.0, 0.2 M Na Thiocyanate.
Cryoprotection: 30s soak in well solution + 12.5% glycerol.
Derivitization: 22 hour soak with KAu(CN)2 added to the drop to a final
concetration of 15 mM
– 2 sites per Assymetric unit, both with 80% occupancy.
Native and Au-MAD data collected to 2.0 A at SSRL BL 9-2
Csa 2 Structure
• Biological units oligomeric state is still unknown
• Tertiary structure is completely unique
• Predicted to function as part of the CASCADE complex in Sulfolobus solfataricus
Brouns et al., Science 321:960
Current functional studies: Csa 2
• What is the oligomeric state of the csa 2 biological unit?
- Analytical Ultracentrifugation
- Sedimentation Velocity to determine shape and diameter
- Sedimentation Equilibrium to determine molar mass
- Multi-Angle Light Scattering
- Molar mass, molecular diameter determination
• Does csa 2 need a particular metal or element to function?
- Inductively coupled plasma mass spectroscopy
- Metal identification and analysis
• Does csa 2 bind to other protein partners or genomic
elements within Sulfolobus solfataricus?
- Liquid Chromatography MS/MS
- Identification of protein-protein interactions within native Sulfolobus solfataricus
Csa 3
Crystallization and Structure Determination
Selenomethionine incorporated protein expressed (10 mg/L of culture) in
BL21(DE3) pLysS grown 30 hours in PASM-5025 (Studier) at 37 C.
Crystals grown (P212121, 2 chains per assymetric unit, 43 % solvent content)
using 4ul + 4ul Sitting drop vapor diffusion with 37.5-42.5% MPD, 5% PEG8000 and 0.1 M Na Cacodylate pH 7.0-7.5.
MAD data Collected at Se Edge at SSRL BL9-1.
Csa 3 structure
Specific transcription factor for transcription of the CRISPR Locus (Koonin)
Biological structure comprised of 2 asymmetric units
Winged helix turn helix DNA binding domain
Contains modified Rossmann fold
Functional Prediction from
Structural Study
• Localization of positive charge within
Rossmann fold reinforces DNA binding
Current Functional Studies: Csa3
• Current model for DNA binding not favorable due to
structural incompatibility
– SAXS small angle x-ray scattering
- Determine the structure of protein in solution
• Does csa 3 bind to other protein partners or genomic
elements within Sulfolobus solfataricus?
– Liquid Chromatography MS/MS
- Identification of protein-protein interactions within
native Sulfolobus solfataricus
Csa 1
• Expresses at high efficiency in Escherichia
coli but does not crystallize
– Metal affinity tag may have been interfering
• Cloned through the Gateway system
excluding affinity tag
• Cloning with C-terminus affinity tag
• Expression trials possible
Csa 1
Csa 1
Cas 5
• This protein has been cloned into an E. coli Expression
vector under the control of the T-7 viral promoter with an
N-terminal 6x Histidine tag using the Gateway system
• Awaiting sequencing results from Nevada Genomics
before proceeding with expression trials
Agarose Gel indicates PCR product is ~820 bp
• Csa 2 and Csa 3
– Structure completely refined
– Pursuing functional studies
• Csa 1 and Cas 5
– Modified constructs entirely cloned
– Transformed into BL21DE3 pLyse S
expression cell line
• CRISPR-cas is a newly discovered
adaptive immune system that evolves at a
high rate to counter phage infection
• CRISPR-associated proteins are key in
proposed mechanism
– Viral DNA degradation
– Incorporation of foreign DNA into spacer
• Nathanael Lintner (Doctoral Candidate)
Derivative crystal optimization
Data collection SSRL beam line 9-1
Structure refinement csa 2, csa 3
Experiment Design csa 2, csa 3
Lawrence Lab members
Robin Gerlach
Thermal Biology Institute, Montana State University
Stanford Synchrotron Radiation Labs and Beam Line 9-1
Lawrence Berkeley National Laboratory
• Funding