Bueno-Carrasco et al.

, 2022

Gupta-Van Duyne Lab Meeting 08/23/2023

Presented by Alexander Kang
 Introduction:

● Tyrosine Hydroxylase (TH) synthesizes

catecholamines (CA)
○ L-Tyr → L-Dopa (First & Rate-limiting step)
○ Epinephrine, Norepinephrine, Dopamine
(DA)
○ motor control, emotion, reward,
biorhythms, learning
● TH mutations lead to neurological
disorders (congenital TH deficiency)
○ Parkinson’s → degrades substantia nigra
○ L-dopa responsive dystonia (DRD)
○ Tremors, postural instability, rigidness
 Bueno-Carrasco et al., 2022
Research Objective:

● Obtain full-length human

TH structure
● Investigate how DA acts
as feedback inhibitor
○ Inhibitory & stabilizing
effects
● Reactivating ability from
phosphorylation from
PKAs
 Methodology:
● Cryo-EM
○ Overcome highly flexible
nature of n-terminal
● Expressed/purified
recombinant human TH
● Gel analysis & optimization
(tetrameric peak, smaller
octomeric peak)
● Used TH1 (isoform 1),
TH is not allosterically
regulated
○ Shortest
N-terminus
● Alanine-rich segment
follows 40-49 → high
helical propensity
 Key Findings:

● Ligand-free TH (apo-TH)
● apo-TH forms tetrameric structure
formed by CD/OD (110 Å X 86 Å X
38 Å) ~3.9 Å final
● Dimerized RD separated 15 Å from
CD (40 × 40 × 22 Å)
● MonoRes: non-isotropic resolution
distribution across 3D map (2Å
central, 10Å RD) → dimeric
structure flexibility

Bueno-Carrasco et al., 2022

 Key Findings:
● Ligand-bound TH (TH-DA)
● 4.1 Å final 3D reconstruction
○ Similar resolution analysis as apo-TH using MonoRes
● Extra cylindrical structure → α-helix
protruding from central mass and contacting
the RDs (3.8 Å)
● Used COOT and PHENIX software for atomic
model building and refinement
○ Cut RD side chains for better resolution
○ Can see helical shape in the N-terminus, specifically in
the first 70 residues (NOT seen in apo-TH)
● Modeled 20-residue alpha-helix (39-58 region)
○ Confirmed w/ THNΔ35
● Alpha-helix penetrated & stabilized active site →
tight DA binding/strong TH inhibition. Bueno-Carrasco et al., 2022
 Key Findings:
● B) (left) apo-TH active site,
(middle) TH(DA) minus the
apo-TH, red is the
internalized alpha helix and
the mass (red arrow) is the
DA
● C) Red and Green segment
shows the shift and
rearrangement in the
presence of DA Bueno-Carrasco et al., 2022
 Key Findings:
● A) THNΔ35 w/ DA
● B) Same w/ CD + OD domains of
THNΔ35 (No RD), black arrows
show a helix docked in active site
● C) TH(DA) vs THNΔ35
● D) THS40p Structure
○ * indicates where a helix was
in TH(DA) and THNΔ35 but
NOT here

Bueno-Carrasco et al., 2022

 Key Findings:
● Used cross linking mass spectrometry (XL-MS) to analyze apo-TH/TH(DA)
○ Helps determine DA stabilizing effect/rearrangement to explain helix
● Incubated w/ crosslinker BS3 before trypsin digestion and LC-MS/MS
analysis. The MS/MS data were searched using StavroX, and the results
were filtered at a false discovery rate (FDR) of less than 5%.
● Identified 300 total peptide-spectrum matches (PSMs) corresponding to
apo-TH and 340 PSMs corresponding to TH(DA)
○ represented 128 unique crosslinked peptides for apo-TH and 120 for TH(DA)
● Analyzed first 20 N-terminal residues of TH (most flexible)
○ Crosslink # double in apo-TH than TH(DA) ← subset of apo-TH = higher
N-terminal flexibility
 SRCD Profiles
● A) apo-TH (broken line) and TH(DA)
(continuous line)
● B) apo-TH (black line), the deletion mutants
THNΔ35 (pink line), THNΔ43 (green line) and
THNΔ70 (orange line), and TH with
phosphorylated S40 (THS40p; blue line)
● C, D) Differential Scanning Calorimetry (DSC)
profiles in absence (broken line) and presence
(continuous line) of DA
○ support the role of the α-helix
sustaining DA-dependent stabilization
● E) Relative TH activity versus DA
concentration for apo-TH (black dots), the
deletion mutants THNΔ35 (pink dots), THNΔ43
(green dots), THNΔ70 (orange dots), and
THS40p (blue dots) Bueno-Carrasco et al., 2022
● F) IC50 (DA amt to inhibit 50% of TH)
 Key Findings:
● S40 phosphorylated TH (THS40p)
○ Phosphorylation at S/T (T8,
S19, S31, S40)
○ Increased IC50 for DA (12.4 ±
2.3 μM), 25-fold higher than
unphosphorylated TH
● Decreased affinity bc helix is
separated from iron-coordinated
DA in active site, rather than
disruption of the helical structure
● Inhibition competes w/ BH4
Incubation
Bueno-Carrasco et al., 2022
● A) MD simulation of S40 phosphorylation
on the interaction between the N-terminal
α-helix and DA
○ slight shift in the position of the N-terminal α-helix
● B) TH(DA) active site (orange α-helix)
● C) Reside interactions between N-terminal
α-helix and adjacent regions in the active
site.
 Discussion

● Compared TH Cryo-EM model and crystallographic structure

○ helical and loop structure in 176-196 vs shifted loop bc of DA
● TH core resolutions range from 2-4 Å, while in PAH it is about 5 Å
○ Polar residues in the PAH OD may allow for different conformations and dimer-tetramer
equilibrium
● Tight DA binding is explained by stabilization of the 39-58α-helix
into the active site
● DA Catechol interacts with Fe(III) ion and specific residues in the
active site, while also establishing interactions with L41 →
immobilizes helix
 Significance & Conclusion

● Better understand inhibitory and stabilizing role of

DA in the regulation of TH activity and protein
turnover
● Develop therapeutic strategies for neurological
disorders associated with deficient TH activity.
● Investigate the TH dephosphorylation reaction
● Explore other post-translational modifications
(acetylation, methylation, etc.)
 Thank You!
Any Questions?