# Presentation: 222522

## Combined X-ray diffraction and neutron scattering study of Aldose reductase  

**Combined X-ray diffraction and neutron scattering study of Aldose reductase  **

- ACA – 2007, Salt Lake city

**Pavel Afonine**

## The project

- 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)

- _Multiple groups collaboration:_

## 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.

- Glucose
- (open form)

- Aldose
- Reductase

- NADPH

- NADP+

- NADP+   NADPH

- Sorbitol
- Dehydrogenase

- Fructose

- Hexoquinase

- Glucose 6-P

- CO2

- NAD+     NADH

- Sorbitol

- LINKED TO DIABETIC COMPLICATIONS

## 3D structure

- NADPH

- 

- α helix

- β sheet

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

## 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.

## Experimental data

- 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)

-   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:

## 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 A&M University_
- 	Tom Ioerger, Jim Sacchettini, Erik McKee (TEXTAL)

## phenix.refine: single program for a very broad range of resolutions

- - Group ADP refinement
- - Rigid body refinement
- - Torsion Angle dynamics

- - Restrained refinement    (xyz, iso / aniso ADP)
- - Automatic water picking

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

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

-           Low                       Middle and High              Subatomic

## 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 didn’t exist at that point). IAS modeling has been performed with PHENIX.
- ...  Let’s have a look at maps.

## X-Rays at 0.66 Å resolution show single protonation of His 110:

- X-Rays at 0.66 Å resolution show single protonation of His 110:

- The information about protonation state of His110 (single or double) is important in understanding how AR binds inhibitors.

## Protonation state of Lys77

- Lys 77

- Tyr 48

- Asp 43

- 1.27

- 1.24

- 3.12

- 2.66

- 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).

## Protonation state of Lys77

- Asp 4 3

- NADP+

- Lys77

- Tyr 48

- 1.25

- 1.28

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

- 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.

## Neutron and joint X/N refinement in PHENIX

- _Macromolecular Neutron Crystallography Consortium (MNC)_

- Lawrence Berkeley National Lab (LBNL)
*Paul Adams*, Pavel Afonine

- Los Alamos National Laboratory
*Paul Langan*, Marat Mustyakimov, Benno Schoenborn

## 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,
- ...)

- Files for COOT, O, PyMol

- Repeated several times

## Refinement statistics

| R-factors, % | Individual
(X-ray or neutron) | Joint (X-ray + neutron) |
| --- | --- | --- |
| Rx | 14 | 13 |
| Rx_free | 18 | 17 |
| Rn | 26 | 26 |
| Rn_free | 32 | 29 |

-    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 gradient’s ratios (similar to CNS)
-        wXN – grid search optimizing Rfree

## Maps: X-ray vs neutron

-  Different maps / techniques – different information

**2mFo-DFc, neutron 2.2 Å (blue), X-ray 1.8 Å (red)**

- Pro 261 (2σ contour)

- Tyr 48 (1.1σ contour)

## Map comparisons: individual vs joint refinement

- Refinement (neutron data only)

**2mFo-DFc, neutron data, 2****σ****, 2.2 Å resolution**

- Refinement (X-ray and neutron data)

- Ser 210

- Arg 255

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

## Lys 77

- The neutron map (red, joint refinement, 2mFo-DFc, 2σ) confirms the observation of the high resolution structure (blue, Fo-Fc map, 2σ) that two of the H-atoms are stronger than the third one.
- (Refinement with phenix.refine)

- Asp 43

- Tyr  48

- NADP+

## 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 -> lower noise -> hopefully we see the protonation state of Lys77

## 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_

***a*** and ***b*** are pre-computed for most of bonds and implemented in PHENIX : better parameterization and refinement.

- Simple gaussian is good enough:

## 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-ray+Neutron 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 

- 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-ray+Neutron 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