Task 9 (MSUD)

Results

Lab journal

For this task we have chosen 5 mutations from HGMD and dbSNP. 2 of them are neutral mutations which do not have functional changes over the protein structure. Following are the 5 mutations:

Database	Accession_code	Mutation	Pathogenic
dbSNP	rs11549938	M82L	False
dbSNP	rs141086188	A222T	False
HGMD	CM045934	C264W	True
HGMD	CM062448	R346H	True
dbSNP	rs61736656	I361V	False

Selection of structure model

The following table shows an overview of all structures that are available for our protein:

Entry	Method	Resolution [Å]	Chain	Positions	R-value	pH	gaps
2BFD	X-ray	1.39	A	46-445	0.15	5.5	289GLY-292SER,293THR-313ASP
2BFF	X-ray	1.46	A	46-445	0.15	5.5	301ARG-305GLU
1X7Y	X-ray	1.57	A	46-445	0.15	5.8	287ARG-313ASP
2BFC	X-ray	1.64	A	46-445	0.144	5.5	288ILE-313ASP
2BFE	X-ray	1.69	A	46-445	0.15	5.5	289GLY-291HIS,293THR-313ASP
1X7Z	X-ray	1.72	A	46-445	0.154	5.8	301ARG-306VAL
1X7W	X-ray	1.73	A	46-445	0.148	5.8	287ARG-313ASP
2BFB	X-ray	1.77	A	46-445	0.145	5.5	287ARG-313ASP
2BEW	X-ray	1.79	A	46-445	0.147	5.5	301ARG-307ASN
1V1R	X-ray	1.80	A	46-445	0.158	5.5	222ASN-231SER,286TYR-314HIS
2BEV	X-ray	1.80	A	46-445	0.139	5.5	301ARG-307ASN
1U5B	X-ray	1.83	A	46-445	0.156	5.8	301ARG-306VAL
1WCI	X-ray	1.84	A	46-445	0.149	5.5	301ARG-309TRP
1OLS	X-ray	1.85	A	46-445	0.172	5.5	292SER-295ASP,298SER-300TYR,301ARG-308TYR
2J9F	X-ray	1.88	A/C	46-445	0.171	5.5	300TYR-313ASP
2BEU	X-ray	1.89	A	46-445	0.171	5.5	301ARG-307ASN
1OLU	X-ray	1.90	A	46-445	0.161	5.5	26ASN-30GLY,287ARG-313ASP
1V16	X-ray	1.90	A	46-445	0.132	5.5	288ILE-313ASP
1V11	X-ray	1.95	A	46-445	0.139	5.5	288ILE-313ASP
1V1M	X-ray	2.00	A	46-445	0.13	5.5	289GLY-313ASP
1X80	X-ray	2.00	A	46-445	0.161	5.8	287ARG-313ASP
1X7X	X-ray	2.10	A	46-445	0.149	5.8	287ARG-313ASP
1OLX	X-ray	2.25	A	46-445	0.161	5.5	301ARG-307ASN
1DTW	X-ray	2.70	A	46-445	0.224	7.5	301ARG-314HIS

Unfortunately all structures contain gaps that span positions 302-304 (corresponding to 347-349 in the reference sequence), so we cannot create a composite structure, that does not contain this gap. We chose 2BFF because it has the smallest gap, a good resolution and a low R-value. It is not resolved at physiological pH, but the only structure with pH 7.5 (1DTW) has a bad resolution. The RMSD between 2BFF and 1DTW is about 0.3, so the different pH does not lead to a different structure and therefore the low pH at that 2BFF was resolved should not be a problem.

Visualization of mutant structures

The mutagen tool of PyMOL was used to introduce mutations to the protein structure. Mutations and their neighboring residues are visualized and shown in following figures.

• Visualization of mutations in BCKDHA (PDB: 2BFF, all residue positions refer to the reference sequence of BCKDHA)
• 

  Local environment of residue 82. Original methionine is replaced by leucine.

• 

  Local environment of residue 222. Original alanine is replaced by threonine.

• 

  Local environment of residue 264. Original cysteine is replaced by tryptophan. Clearly, the mutant residue tryptophan clashes with the binding site (Green) for ion binding.

• 

  Local environment of residue 346. Original arginine is replaced by histidine.

• 

  Local environment of residue 361. Original isoleucine is replaced by valine.

Energy comparisons

FoldX

Mutation	PDB	SD	total	energy	Backbone	Hbond	Sidechain	Hbond	Van der Waals
M82L	pdb2bff_1	0	0.08	0.01	-0.05	0.46	-0.1	0	0.44
A222T	pdb2bff_2	0	1.22	0.17	-1.17	-0.7	0.02	1.48	-0.51
C264W	pdb2bff_3	0	34.77	-0.16	-0.15	-1.99	-0.09	2.88	-2.95
R346H	pdb2bff_4	0	1.55	1.28	1.9	1.36	-0.57	-3.03	0.73
I361V	pdb2bff_5	0	0.78	-0.02	0.01	0.54	-0.02	-0.29	1.06

Resulted structural models were compared to the structures calculated by SCWRL. By using alignment tool of PyMOL, we did not find any global deviation between the structures. Sequentially, structures produced by SCWRL have some residues missing. Following table shows the sequential difference.

Mutation	RMSD(Å)	Missing residues in SCWRL
M82L	0.0	SER76,HIS96,MET146,LEU325,SER326
A222T	0.0	SER76,HIS96,MET146,LEU325,SER326
C264W	0.0	SER76,HIS96,MET146,LEU325,SER326
R346H	0.0	SER76,HIS96,MET146,LEU325,SER326
I361V	0.0	SER76,HIS96,MET146,LEU325,SER326

In 3 mutant structures the mutation residues have different side-chain conformation between results of FoldX and SCWRL. The other 2 mutant structures do not show such difference.

• Mutation residues with different side-chain conformations (FoldX in red, SCWRL in blue)
• 

  Slightly difference between side-chain conformations of LEU82 in mutant M82L(FoldX in red, SCWRL in blue)

• 

  Difference between side-chain conformations of TRP264 in mutant C264W(FoldX in red, SCWRL in blue). They show completely different orientation of the heterocyclic ring in tryptophane.

• 

  Slightly difference between side-chain conformations of HIS346 in mutant R346H(FoldX in red, SCWRL in blue)

Minimise

The enegies of each Minimise run for the wild type and mutant structures are given in the following table. For the mutant structures there was only an output for structures calculated with FoldX. For input structures from SCWRL, Minimise gave no output (probably there was a problem with gaps).

run	1	2	3	4	5
WT	-17591	-17424	-16955	-16792	-16578
M82L FoldX	-17981	-17722	-17298	-17001	-16809
A222T FoldX	-17940	-17708	-17297	-17001	-16800
C264W FoldX	-15606	-17724	-17319	-17164	-16897
R346H FoldX	-18014	-17769	-17339	-17049	-16853
I361V FoldX	-18010	-17684	-17313	-17012	-16799

A reduction in the energy can only be observed for C264W, and only for the second recursive run. All other energies increase slightly with every run, but are overall similar to each other.

Discussion

• For the disease causing mutations (C264W and R346H), FoldX and SCWRL come to different results regarding the side chain conformation. This gives an indication, that the substituted amino acids do not fit into the local environment compared to the wild type structure. So they change the structure of the protein and hence can alter its function.
• The tryptophane that replaces a cysteine in C264W reaches into an ion binding site and so might interfere with binding of that ion, resulting in inhibition of enzymatic activity.
• We did not observe any minimization when running Minimise on the WT and mutant structures (except for C264W), the energy rather stagnated around a value of about -17000. Probably the structures are already in an energy minimum and can not be minimized any more, which does not mean that the mutant structures keep the functionality of the wild type.