A Conserved Threonine Spring-loads Precursor for Intein Splicing
Albert K. Dearden1, Brian Callahan2, Patrick Van Roey3, Zhong Li3, Utsav Kumar1, Marlene Belfort2,**,
and Saroj K. Nayak1,**
1
Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180, United
States
2
Department of Biological Sciences, University at Albany, Albany, New York 12222, United States
3
Wadsworth Center, New York State Department of Health, Albany, New York 12201, United States
**
Corresponding authors: nayaks@rpi.edu, mbelfort@albany.edu
Supplemental Material
Table S1. Gly-1 dihedral angles and their corresponding location in the Ramachandran plot. Values obtained for the various mutations. The first two mutations were derived experimentally whereas
the remaining data are from computational work. Thr QM and Thr QM/MM refer to the full quantum
mechanical calculation and the mixed quantum mechanical and molecular mechanical methods respectively, while Thr-S refers to the threonine calculation starting from the completed serine optimization.
Region indicates the location in the Ramachandran plot to which the dihedral angles correspond, either
allowed (A) or non-allowed (NA).
Mutation

ψ
Region
Thr69 Exp.
-122.5
86.9
NA
Ala Exp.
-88.0
29.8
A
Thr QM
-134.2
74.7
NA
Thr QM/MM
-123.2
77.8
NA
Thr-S
-124.6
53.0
A
Gly
-99.4
54.4
A
Ala
-119.6
50.7
A
Ser(1)
-124.6
53.0
A
Ser(2)
-129.6
72.0
NA
Val
-116.9
51.9
A
Cys
-119.7
52.4
A
Asp
-117.6
51.9
A
1
Table S2. Computational Values for the Reaction Barriers. Computational values for the reaction
barriers involving the native Thr69 and the T69A mutation. Each mutation is grouped with its corresponding allowed (A) or non-allowed (NA) region in the Ramachandran plot, its energy barrier, and
charge values. HThr refers to the hydroxyl hydrogen of the threonine side chain, S Cys to the attacking
sulfur of the cysteine side chain, OGly to the glycine oxygen receiving the hydrogen during the transition,
OPro to the carbonyl oxygen atom of Pro-2, and NCys to the peptide nitrogen at the glycine-cysteine junction.

ψ
Region
Barrier
(kcal/mol)
Charge (e)
HThr
SCys
OGly
OPro
NCys
-123.2
77.8
NA
14.8
0.52
-0.26
-0.67
-0.71
-0.64
-124.6
53.0
A
34.6
0.52
-0.37
-0.65
-0.70
-0.62
-119.6
50.7
A
24.9
N/A
-0.51
-0.65
-0.67
-0.60
-123.2
77.8
NA
48.3
N/A
-0.25
-0.77
-0.68
-0.60
Thr
Ala
Table S3. Oligonucleotides used in this study
Gene/Purpose
ID
Sequence
Site
Feature
DnaE / T69A 2845
mutation
GTTCAGTAATCCGAGCTGCGTCTG
ACCACCGCTTTTTAAC
-
C-term
fragment
DnaE / T69A 2846
mutation
GTTAAAAAGCGGTGGTCAGACGCA GCTCGGATTACTGAAC
N-term
fragment
DnaE / T69S 3146
mutation
GATGGTTCAGTAATCCGAGCTAGC
TCTGACCACCGCTTTTTAACCACC
G
-
C-term
fragment
DnaE / T69S 3147
mutation
CGGTGGTTAAAAAGCGGTGGTCAG
AGCTAGCTCGGATTACTGAACCAT
C
-
N-term
fragment
DnaE / T69A 3085
and
T69S
(pET45b)
AAACACGTGCTGGTGCCGCGCGGC
AGTCCGGATCCCTTTTGCCCTGGTT
GCCTGTC
PmlI
LVPRG
protease
site, CPGC
DnaE T69A and 3086
T69S (pET45b)
AAACTCGAGTCACTGGACGTTAAA
GCAGTTAGCTGCGATAGCGCC
XhoI
REV primer
2
Table S4. Data collection, phasing and refinement statistics
Resolution (Å)
29.6-1.8 (1.86-1.8)
Unique reflections
16636
Redundancy
2.56 (2.37)
Completeness (%)
99.3 (98.2)
Rmer (%)
0.034 (0.342)
I/σ(I)
13.6 (2.5)
Rcryst
0.226
Rfree
0.268
R.m.s.d. from ideality
Bond lengths (Å)
0.005
Bond angles ()
1.2
Dihedrals ()
24.0
2
Average B-factors (Å )
Protein
37.0
Solvent
45.9
3
Figure S1. Comparison of reported CPGC loops with the wild type and T69A intein. Other than in the
engineered DnaE inteins discussed here, a Cys-Pro-Gly-Cys peptide with a disulfide bond has been
found in the PDB in only two structures: PDB entries 1JBQ (red) and 1M6Y (orange). Superposition of
these four observations shows that the conformation of this loop in the native DnaE structure (3NZM,
green) deviates more from the two unrelated observations than the loop in the T69A mutant (4GIG, cyan) and that the difference is the greatest in the conformation of Gly-1 (top right).
Figure S2. Superposition of the CPGC loop in the native and T69A mutant of the DnaE intein. The Thr
to Ala mutation, in addition to having removing the strain on the loop at Gly-1 allows for the insertion
of a water molecule between the loop and Ala69 that results in a radically different hydrogen bonding
pattern as discussed in the text.
4