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Combined X-ray diffraction and neutron scattering
study of Aldose reductase
Pavel Afonine
ACA 2007, Salt Lake city
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The project
Multiple groups collaboration
1. IGBMC, France A. Mitschler,F. Ruiz, I.
Hazemann, A. Cousido, T. Petrova, A. Podjarny
2. ILL and EMBL, Grenoble, France. P. Timmins,
M. Blakeley, M. Haertlein, M.T. Dauvergne, F.
Meilleur, D. Myles 3. ABCC, NCI, SAIC and
Universidad de la Republica, Uruguay R. Cachau,
O. Ventura 4. Biosciences Division/Structural
Biology Center, ANL, Argonne, IL,USA A.
Joachimiak, S. Ginell, R. Sanishvili5.
Institute for Diabetes Discovery, Branford, CT,
USA M. Van Zandt 6. Tools for neutron
crystallography (LANL, USA Paul Langan, Marat
Mustyakimov, Benno Schoenborn LBNL, USA Paul
Adams, Pavel Afonine)7. Subatomic resolution
modelling (IGBMC A. Urzhumtsev, IMPB RAS V.
Lunin)
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Aldose Reductase

• Aldose Reductase (AR) is a NADPH-dependent enzyme
  that reduces a wide range of substrates, such as
  aldehydes, aldoses and corticosteroids. As it
  reduces D-glucose into D-sorbitol, it is believed
  to cause severe degenerative complications of
  diabetes (C. Yabe-Nishimura, Pharmacological
  Review 50, 21 (1998)).
• Glucose methabolism through the polyol pathway
  Cellular glucose is phosphorylated into glucose
  6-phosphate by Hexoquinase under normoglycemia
  conditions. Only a minor part of glucose goes
  into an alternative route called the
  polyol-pathway (grey ellipsoid in the figure).
  Aldose is reduced to sorbitol by Aldose Reductase
  and sorbitol converted to fructose by Sorbitol
  dehydrogenase.

Hexoquinase
CO2
Glucose 6-P
NADP NADPH
Glucose (open form)
Aldose Reductase
Sorbitol Dehydrogenase
Fructose
Sorbitol
NADPH
NADP
NAD NADH
LINKED TO DIABETIC COMPLICATIONS
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3D structure
NADPH

a helix
ß sheet
The structure originally was solved from the pig
enzyme (1989) and showed a TIM barrel structure
(conserved fold consisting of 8 a-helices and 8
parallel ß-strands named after
triosephosphateisomerase), with the coenzyme
sitting on top of the barrel.
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Goal

• To fully understand the catalytic mechanism
  and binding of inhibitors, information is
  required about the protonation state of catalytic
  residues, such as His110, Asp43, Lys77 and Tyr48.
  
• Combining analysis of X-ray structures at
  subatomic resolution, structure obtained from
  neutron data and quantum-chemical modeling (not
  discussed here) can provide the complete picture.

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Experimental data

• The nature of proton transfer processes
  combined with the weak X-ray scattering signal
  from hydrogen atoms, which excludes their
  observation at resolutions lower than 1.2 Å.
• Neutron diffraction is particularly efficient
  at locating Deuterium (D) atoms the scattering
  length of D is similar to that of C, N and O
  atoms.
• The extraordinary quality of AR crystals allows
  structural studies at a level of detail not
  available before for an enzyme of this size

• Hydrogenated enzyme (X-ray, 0.66 Å, 100 K)
• Fully deuterated enzyme (X-ray, 0.82 Å, 15 K)
• Fully deuterated enzyme (neutron 2.2 Å and X-ray
  1.8 Å, 293K)

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PHENIX

• Subatomic resolution (proper parameterization
  and refinement)
• Neutron data (use of X-ray and neutron data
  joint X/N refinement)
• PHENIX is right choice
• The PHENIX project is a collaboration between
  several groups to develop tools for automated
  crystallography

• Los Alamos National Lab
• Tom Terwilliger, Li-Wei Hung (SOLVE / RESOLVE)
• Paul Langan, Marat Mustyakimov, Benno Schoenborn
  (Neutron crystallography) (separate funding)
• Cambridge University, UK
• Randy Read, Airlie McCoy (PHASER)
• Duke University Jane David Richardson, Ian
  Davis (MolProbity)
• Lawrence Berkeley National Lab
• Paul Adams, Pavel Afonine, Ralf
  Grosse-Kunstleve, Nigel Moriarty, Peter Zwart
  (CCI APPS)
• Texas AM University
• Tom Ioerger, Jim Sacchettini, Erik McKee (TEXTAL)

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phenix.refine single program for a very broad
range of resolutions
Low Middle and
High Subatomic

• - Bond density
• - Unrestrained refinement
• FFT or direct
• Hydrogens

- Restrained refinement (xyz, iso / aniso
ADP) - Automatic water picking
- Group ADP refinement - Rigid body refinement -
Torsion Angle dynamics

• - Automatic NCS restraints
• - Simulated Annealing
• - Occupancies (individual, group)
• - TLS refinement
• - Twinned data
• X-ray, Neutron, joint X-ray Neutron refinement

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High-Resolution X-ray Studies of Aldose Reductase

• Two X-ray structures analyzed at 0.66 Å and at
  0.82 Å resolution
• Shows single protonation of His110.
• Start seeing protonation state of Lys77, but
  signal close to the noise (need
  cross-validation).
• On-going work new modeling at subatomic
  resolution (IAS) may improve maps and help reveal
  the Lys77 protonation state.
• Both structures were originally refined with
  SHELX (phenix.refine didnt exist at that point).
  IAS modeling has been performed with PHENIX.
• Lets have a look at maps.

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X-Rays at 0.66 Å resolution show single
protonation of His 110
Protonation state of His110
The information about protonation state of His110
(single or double) is important in understanding
how AR binds inhibitors.
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Protonation state of Lys77
Asp 43
Lys 77 makes a salt bridge with Asp 43 through
short H-bond. The structure at 0.66 Å suggests
alternative conformations of the H-atom in this
salt bridge, mainly through the difference in the
C-O bonds in Asp 43, but this is not confirmed by
the H-atom peaks (Shelx refinement).
1.27
1.24
2.66
3.12
Lys 77
Tyr 48
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Protonation state of Lys77
Structures with Fo-Fc map. Deuterated (0.82Å)
blue, hydrogenated (0.66 Å) red. The deuterated
structure also shows the difference in bond
lengths indicating protonation of Asp 43
however in this case the map shows the proton
on the side of Asp 43 !! (Shelx
refinement) Peak height close to noise.
Asp 4 3
1.25
1.28
NADP
Lys77
Tyr 48

• Two possibilities to try to confirm the position
  of this H neutrons or IAS.

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Neutron and joint X/N refinement in PHENIX
Macromolecular Neutron Crystallography Consortium
(MNC)
Los Alamos National Laboratory Paul Langan, Marat
Mustyakimov, Benno Schoenborn
Lawrence Berkeley National Lab (LBNL) Paul Adams,
Pavel Afonine
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Neutron and joint X/N refinement in PHENIX
Input data and model processing Refinement
strategy selection Bulk-solvent, Anisotropic
scaling, Twinning parameters refinement Ordered
solvent (add / remove) Target weights
calculation Coordinate refinement (rigid body,
individual) (minimization or
Simulated Annealing) ADP refinement (TLS, group,
individual iso / aniso) Occupancy refinement
(individual, group) Output Refined model,
various maps, structure factors, complete
statistics
PDB model, Any data format (CNS, Shelx, MTZ, )
Repeated several times
Files for COOT, O, PyMol
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Refinement statistics

• X-ray data 1.8 Å, neutron data 2.2 Å, data
  collected at room termerature
• Refinement strategy individual ADP
  coordinates, automatic water picking
• Target used for joint X-ray neutron
  refinement
• TargetJOINT EXRAY wXC ENEUTRON wNC wXN
  EGEOM
• wXC, wNC determined as gradients ratios
  (similar to CNS)
• wXN grid search optimizing Rfree

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Maps X-ray vs neutron
2mFo-DFc, neutron 2.2 Å (blue), X-ray 1.8 Å (red)
Pro 261 (2s contour)
Tyr 48 (1.1s contour)

• Different maps / techniques different
  information

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Map comparisons individual vs joint refinement
2mFo-DFc, neutron data, 2s, 2.2 Å resolution
Refinement (neutron data only)
Refinement (X-ray and neutron data)
Arg 255
Ser 210

• Neutron maps are improved after joint refinement
  comparing to refinement with neutron data only

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Protonation of Lys 77
The neutron map (red, joint refinement, 2mFo-DFc,
2s) confirms the observation of the high
resolution structure (blue, Fo-Fc map, 2s) that
two of the H-atoms are stronger than the third
one. (Refinement with phenix.refine)
Asp 43
NADP
Tyr 48
Lys 77
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High-Resolution X-ray Studies Bond densities and
H atoms
Red Contours Fo-Fc 0.28 e/A3
Blue Contours 2Fo-Fc 3.85 e/A3
Hypothesis to check (on-going work) if we model
IAS -gt lower noise -gt hopefully we see the
protonation state of Lys77
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Modeling at subatomic resolution IAS model

• Basics of IAS model
• Afonine et al, Acta Cryst. D60, 2004
• First examples of use in PHENIX
• Afonine et al, (submitted to Acta Cryst D)

IAS modeling in PHENIX
Simple gaussian is good enough
a and b are pre-computed for most of bonds and
implemented in PHENIX better parameterization
and refinement.
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Conclusions

• Ultra high resolution structure determination
  provides very detailed description of the ordered
  part of protein structures
• Neutron diffraction structure determination can
  improve the hydrogen visibility obtained by X-Ray
  ultra high resolution structures.
• Joint X-rayNeutron structure and subatomic
  resolution structure
• Confirms protonation states observed by subatomic
  X-Ray studies
• Suggests proton mobility between Asp 43 and Lys
  77
• Shows change of the Lys 77-Tyr 48 distance

• Funding
• NIH / NIGMS P01GM063210, R01GM071939,
  P01GM064692
• LBNL DE-AC03-76SF00098
• PHENIX industrial consortium