Group meeting Mahavir Singh 01/31/2012. Part I Tetrahymena telomerase holoenzyme core protein p65 Part II Human Shq1, an H/ACA RNP assembly protein

Tetrahymena telomerase holoenzyme assembly Part I: Tetrahymena telomerase assembly protein p65 Min B and Collins K, Mol Cell, 2009 p50 TER p65 TERT Catalytic core components

Structural basis for telomerase RNA recognition and RNP assembly by the holoenzyme La family protein p65 Mahavir Singh, Zhonghua Wang, Bon-Kyung Koo, Anooj Patel, Duilio Cascio, Kathleen Collins ‡, and Juli Feigon* Previous/current results Re-re-submitted on 12/26/2011! Under review

Previous results: NMR and X-ray crystal structure of p65 C-terminal domain NMR structure C2ΔL1 ( Δ ) X-ray crystal structure C1ΔL2 ( Δ ) NMR structure X-ray crystal structure

Previous results: X-ray crystal structure of p65-Cter and TER S4 RNA complex X-ray crystal structure of p65-Cter and TER S4 complex C1ΔL2 ( Δ )/TER S4

p65 TERT Model for assembly of Tetrahymena telomerase catalytic core TERp65TERT

Question # 1 Does β2-β3 loop play a role in RNA binding and conformation change?

Does β2-β3 loop play a role in RNA binding? Background and next C1C1ΔL2 (Δ ) C1ΔL3 (Δ ) C1ΔL4 (Δ ) C1ΔL1 (Δ ) Interaction with S4 RNA (nM) 36.4± ±7.1101±21.2?? Interaction* with TER FL (nM) ~0.6~0.7~2?? Effect on catalytic core assembly** +++?? * Full length p65 was used in the experiment. ** Full length p65 and TERT (1-516) were used in these experiments.

C1∆L3 (Δ ) and C1∆L4 (Δ ) remain folded: 2D HSQC NMR C1ΔL3 (Δ ) C1ΔL4 (Δ ) C1 C1ΔL3 or C1ΔL3 Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM MHZ 600 MHz

C1∆L3 (Δ ) and C1∆L4 (Δ ) vs, S4 RNA: ITC results C1ΔL3 (Δ ) C1ΔL4 (Δ ) Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM 25 deg

Does flexible β2-β3 loop play a role in RNA binding? Background and next C1C1ΔL2 (Δ ) C1ΔL3 (Δ ) C1ΔL4 (Δ ) C1ΔL1 (Δ ) Interaction with S4 RNA (nM) 36.4± ±7.1101± ± ±7.8 Interaction* with TER FL (nM) ~0.6~0.7~2nr*** Effect on catalytic core assembly** +++nr*** * Full length p65 was used in the experiment. ** Full length p65 and TERT (1-516) were used in these experiments. *** Not required Conclusions: Loop does not play a role in RNA binding and catalytic core assembly. Minor changes in K D s are due to some insignificant changes in structure because of loop deletion Moreover, complete loop has a net theoretical neutral charge at physiological pH Loop in C2ΔL1 (NMR)

Model of RNA recognition and conformational change by p65-Cterminal RRM2 (GA-bulge recognition model) Importance of β-sheet or α3-helix for RNA interaction and conformational change

Effect of mutation of conserved residues Y407 and R465 on RNA binding: background C1 α3α3x * C2 α3 C1ΔL2 (Δ ) α3α3x *

Question # 2 Are both β-sheet or α3-helix important for RNA binding and conformational change?

Interaction with S4 RNA (nM) 36.4±3.6101±21.2>1mM No binding ???? Interaction* with TER FL (nM) ~0.6~2~20???? Effect on catalytic core assembly** ++-???? * Full length p65 was used in the experiment. ** Full length p65 and TERT (1-516) were used in these experiments. C1 α3α3x C2 α3 C1ΔL2 (Δ ) α3α3x Importance of β-sheet or α3-helix for RNA interaction and conformational change Conclusions so far: 1.α3x is absolutely required for p65-Cter and minimal S4 interaction. 2.Deletion of α3x in p65-FL decrcreas its interaction with TER FL by ~ 40 fold. 3.Deletion of α3x in p65-FL abrogates the catalytic core (p65-TER-TERT) assembly. C1 α3α3x * Y407A C1ΔL2 (Δ ) α3α3x * Y407A C1 α3α3x * R465A C1ΔL2 (Δ ) α3α3x * R465A

Y407A and R465A mutation on p65-C1 do not effect the folding of the domain C1 α3α3x * Y407A C1 α3α3x * R465A C1 C1-Y407A or C1-R465A Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM DTT pH MHZ 600 MHz Note: CD scan and melting results not shown

Y407A and R465A mutation on p65-C1 decreases its interaction with S4 RNA significantly Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM 25 deg C1 α3α3x * Y407A C1 α3α3x * R465A

Y407A and R465A mutation on p65-C1ΔL2 (crystal construct) do not effect the folding of the domain C1 C1ΔL2-Y407A or C1ΔL2-R465A Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM DTT pH MHZ 600 MHz Note: CD scan and melting results not shown C1ΔL2 (Δ ) α3α3x * R465A C1ΔL2 (Δ ) α3α3x * Y407A

Condition: 20 mM NaH2PO4 50 mM NaCl 1 mM 25 deg C1ΔL2 (Δ ) α3α3x * R465A C1ΔL2 (Δ ) α3α3x * Y407A Y407A and R465A mutation on p65-C1ΔL2 (crystal construct) decreases its interaction with S4 RNA significantly

Interaction with S4 RNA (nM) 36.4±3.6101±21.2>1mM No binding 389.1± ± ± ±46.8 Interaction* with TER FL (nM) ~0.6~2~20???? Effect on catalytic core assembly** ++-???? * Full length p65 was used in the experiment. ** Full length p65 and TERT (1-516) were used in these experiments. C1 α3α3x C1 α3α3x * C2 α3 C1ΔL2 (Δ ) α3α3x C1ΔL2 (Δ ) α3α3x * C1ΔL2 (Δ ) α3α3x * C1 α3α3x * Importance of β-sheet or α3-helix for RNA interaction and conformational change Conclusions so far: 1.α3x is absolutely required for p65-Cter and minimal S4 interaction. 2.Deletion of α3x in p65-FL decrcraes its interaction with TER FL by ~ 40 fold. 3.Deletion of α3x in p65-FL abrogates the catalytic core (p65-TER-TERT) assembly. 4.Mutation of conserved residue, Y407 of newly identified RNP3 motif, involved in recognition of G of SL4 GA bulge, (Y407A) in p65-Cter have significant effect on S4 binding (K D by ~ 6-7 times). 5.Mutation of conserved residue, R465 involved in recognition of both G and A of SL4 GA bulge, (Y407A) in p65- Cter have significant effect on S4 binding (K D by ~ 14 times). Y407A R465A

Question # 3 Is there a correlation between NMR and CD spectroscopy insights and protein crystallizibility? a tale of crystallization of p65 C-terminal domain

Thermal stability measurement of p65 C-terminal constructs using CD spectroscopy: finding the most stable construct ConstructTm (°C) (C1)55.5

NMR het-noe a measure of protein backbone flexibility (previous results) C2 (Δ ) α3 C2ΔL1 (Δ ) α3 C2ΔL3 (Δ ) α3 NMR structure of p65-C2ΔL1 Conclusion: The loop residues in different constructs remain flexible, hence probably hinder crystallization! Check the crystal construct to show that all the loop residues was critical to get crystals.

Thermal stability measurement of new p65 C-terminal constructs designed after deleting the flexible loop, using CD spectroscopy ConstructTm (°C) (C1)55.5 C1Δ C1Δ C1Δ

Thermal stability measurement of new p65 C-terminal constructs designed after deleting the flexible loop, using CD spectroscopy: finding the most stable! ConstructTm (°C) (C1)55.5 C1Δ C1Δ C1Δ C1Δ

Thermal stability measurement of new p65 C-terminal constructs: the most stable construct crystallizes ConstructTm (°C) (C1)55.5 C1Δ C1Δ C1Δ C1Δ

1.p65 C-terminal β2-β3 loop, conserved residue of β-sheet (Y407 and R465), and α3-helix. Catalytic core assembly assays of these mutants in p65-FL. May be these results will go in current paper, otherwise write a manuscript. 2.CD, NMR, and crystallization. Finish het-noe experiment on crystal construct (C1ΔL2) Write a small method/observation paper 3.New p65-LRC constructs based on p65-RRM2 and p65:TER model structure (LRCΔL2). 4.Investigate the role of p65-N terminal domain? Next >>>>

Biogenesis and assembly of eukaryotic H/ACA snoRNP – role of Shq1? Part II: Shq1, a H/ACA snoRNP assembly factor 28 Singh M e. al. Structure and functional studies of the CS domain of the essential H/ACA ribonucleoparticle assembly protein SHQ1. J Biol Chem Jan 16;284(3): Grozdanov PN et. al. SHQ1 is required prior to NAF1 for assembly of H/ACA small nucleolar and telomerase RNPs. RNA Jun;15(6): Godin KS et. al. The box H/ACA snoRNP assembly factor Shq1p is a chaperone protein homologous to Hsp90 cochaperones that binds to the Cbf5p enzyme. J Mol Biol Jul 10;390(2): Li S et. al. Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita. EMBO J.2011, 30(24): Walbott H et. al. The H/ACA RNP assembly factor SHQ1 functions as an RNA mimic. Genes Dev. 2011, 25(22): yShq1 CS domainyShq1 SSD domain ‘maki fold’

Human Shq1, introduction and why we studying CS domain: background Li S et. al. Structure of the Shq1-Cbf5-Nop10-Gar1 complex and implications for H/ACA RNP biogenesis and dyskeratosis congenita. EMBO J.2011, 30(24): Yeast H/ACA complex (without RNA and Nhp2) Taylor BS et. al. Integrative genomic profiling of human prostate cancer. Cancer Cell Jul 13;18(1):11-22 Human Shq1 is found mutated in prostate cancer (P22S confirmed somatic mutation)

Specific aims What is the effect of P22S mutation on CS domain structure? Structure of human Shq1 FL (CS+SSD) protein?

Human Shq1 CS-WT and CS-P22S behave as dimer - tetramer (or monomer – dimer) on gel filtration column?? 577 First purification on lab S75 column Purification on SEC-MALS column CS-WT Expected Mw: kDa CS-P22S Expected Mw: kDa CS-WT CS-P22S 17.5 kDa 44 kDa 17.5 kDa 44 kDa CS-WT CS-P22S

SEC-MALS brief introduction and lesson learned -Molecular weight -Dispersity

CS-WT and CS-P22S for monomer with a fraction as dimer: results from SEC-MALS Result after SEC-MALS: Mw (kDa)12.6 (±8.7%) Polydispersity1.008 (±12.2%) Result after SEC-MALS: Peak 1Peak 2 Mw (kDa)13.6 (±8.0%)24.0 (±6.1%) Polydispersity1.035 (±11.3%)1.016 (±8.2%) CS-WT Expected Mw: kDa CS-P22S Expected Mw: kDa Peak 1 Peak 2

NMR HSQC spectra of CS-WT and CS-P22S show differences in peak pattern: (previous results) hCS-WT hCS-P22S Condition: 20 mM NaH2PO4 100 mM NaCl 1 mM DTT pH MHZ 600 MHz

Crystal structure of human CS-WT domain at 1.9Å resolutions Human CS-WT Yeast CS-WT Loop 1 (β2-β3) Pro22 C N β2 β1 β3 β4 β5 β6 β7 α1 Loop 1 (β2-β3) Pro22

The His-tag in CS-WT construct is involved in domain swap? N C N 1.Artifact of crystallization. 2.Cut the his-tag. 3.Analyze crystal from other condition.

Structural basis of P22S mutation: can we tell something? P22-Y23 P58-Y59 Human vs. yeast CS domain Loop 1 (β2-β3) Pro22

Physical characterization of CS-WT and CS-P22S: CD melting experiment show that P22S mutation destabilizes the protein ConstructTm (°C) hCS-WT59 hCS-P22S50

Human Shq1 constructs (CS+shq1 domain) Results: No crystals

1.Try to solve hCS-P22S structure or do chemical shift mapping (with ZW) to find difference between CS-WT vs. CS-P22S. 2.Re-do SEC-MALS to reconfirm the state of hCS-WT and hCS-P22S mutant monomer-dimer equillibrium. 3.Write a manuscript on the results. 4.Crystallization trials for full length human Shq1. Contd! 5.Express and purify two new constructs of TCAB1! Next >>>>