Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and ...size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis–trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5′-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
Fluorine atoms are known to display scalar 19F–19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that ...fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F–19F (TS J FF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3 groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F–19F TOCSY experiments are sufficiently sensitive to detect TS J FF couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative J evolution experiment enables the measurement of TS J FF coupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N 6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F–19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F–19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances.
Fluorine atoms are known to display scalar
F-
F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that ...fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space
F-
F (
) couplings even if the
F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF
groups,
-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that
F-
F TOCSY experiments are sufficiently sensitive to detect
couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative
evolution experiment enables the measurement of
coupling constants that are up to five times smaller than the
F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of
-(trifluoroacetyl)-l-lysine (TFA-Lys) and
F-
F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding,
F-
F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of
H NMR resonances.
Extranodal natural killer T-cell lymphoma (NKTCL; nasal type) is an aggressive malignancy with a particularly high prevalence in Asian and Latin American populations. Epstein-Barr virus infection has ...a role in the pathogenesis of NKTCL, and HLA-DPB1 variants are risk factors for the disease. We aimed to identify additional novel genetic variants affecting risk of NKTCL.
We did a genome-wide association study of NKTCL in multiple populations from east Asia. We recruited a discovery cohort of 700 cases with NKTCL and 7752 controls without NKTCL of Han Chinese ancestry from 19 centres in southern, central, and northern regions of China, and four independent replication samples including 717 cases and 12 650 controls. Three of these independent samples (451 cases and 5301 controls) were from eight centres in the same regions of southern, central, and northern China, and the fourth (266 cases and 7349 controls) was from 11 centres in Hong Kong, Taiwan, Singapore, and South Korea. All cases had primary NKTCL that was confirmed histopathologically, and matching with controls was based on geographical region and self-reported ancestry. Logistic regression analysis was done independently by geographical regions, followed by fixed-effect meta-analyses, to identify susceptibility loci. Bioinformatic approaches, including expression quantitative trait loci, binding motif and transcriptome analyses, and biological experiments were done to fine-map and explore the functional relevance of genome-wide association loci to the development of NKTCL.
Genetic data were gathered between Jan 1, 2008, and Jan 23, 2019. Meta-analysis of all samples (a total of 1417 cases and 20 402 controls) identified two novel loci significantly associated with NKTCL: IL18RAP on 2q12.1 (rs13015714; p=2·83 × 10−16; odds ratio 1·39 95% CI 1·28–1·50) and HLA-DRB1 on 6p21.3 (rs9271588; 9·35 × 10−26 1·53 1·41–1·65). Fine-mapping and experimental analyses showed that rs1420106 at the promoter of IL18RAP was highly correlated with rs13015714, and the rs1420106-A risk variant had an upregulatory effect on IL18RAP expression. Cell growth assays in two NKTCL cell lines (YT and SNK-6 cells) showed that knockdown of IL18RAP inhibited cell proliferation by cell cycle arrest in NKTCL cells. Haplotype association analysis showed that haplotype 47F-67I was associated with reduced risk of NKTCL, whereas 47Y-67L was associated with increased risk of NKTCL. These two positions are component parts of the peptide-binding pocket 7 (P7) of the HLA-DR heterodimer, suggesting that these alterations might account for the association at HLA-DRB1, independent of the previously reported HLA-DPB1 variants.
Our findings provide new insights into the development of NKTCL by showing the importance of inflammation and immune regulation through the IL18–IL18RAP axis and antigen presentation involving HLA-DRB1, which might help to identify potential therapeutic targets. Taken in combination with additional genetic and other risk factors, our results could potentially be used to stratify people at high risk of NKTCL for targeted prevention.
Guangdong Innovative and Entrepreneurial Research Team Program, National Natural Science Foundation of China, National Program for Support of Top-Notch Young Professionals, Chang Jiang Scholars Program, Singapore Ministry of Health's National Medical Research Council, Tanoto Foundation, National Research Foundation Singapore, Chang Gung Memorial Hospital, Recruitment Program for Young Professionals of China, First Affiliated Hospital and Army Medical University, US National Institutes of Health, and US National Cancer Institute.
Abstract
C
22
H
50
Ag
2
N
2
P
2
S
4
, triclinic,
P
1̄ (no. 2),
a
= 9.0672(2) Å,
b
= 11.2091(3) Å,
c
= 16.6853(4) Å,
α
= 91.097(2)°,
β
= 90.363(2)°,
γ
= 110.989(2)°,
V
= 1582.85(7) Å
3
,
Z
= 2,
R
gt
(
...F
) = 0.0241,
wR
ref
(
F
2
) = 0.0653,
T
= 100(2) K.
Abstract
C
22
H
46
Ag
2
N
2
P
2
S
4
, triclinic,
P
1̄ (no. 2),
a
= 10.2008(3) Å,
b
= 12.2058(3) Å,
c
= 13.2466(4) Å,
α
= 88.155(2)°,
β
= 87.256(2)°,
γ
= 69.324(2)°,
V
= 1541.12(8) Å
3
,
Z
= 2,
R
gt
(
...F
) = 0.0237,
wR
ref
(
F
2
) = 0.0621,
T
= 100(2) K.
Abstract
C
46
H
82
Cu
2
N
2
P
2
S
4
, triclinic,
P
1̄ (no. 2),
a
= 11.6189(2) Å,
b
= 12.2846(2) Å,
c
= 18.1744(2) Å,
α
= 97.3210(10)°,
β
= 106.3080(10)°,
γ
= 99.312(2)°,
V
= 2415.65(7) Å
3
,
Z
= 2,
R
...gt
(
F
) = 0.025,
wR
ref
(
F
2
) = 0.066,
T
= 100(2) K.
Abstract
C
46
H
86
Cu
2
N
2
P
2
S
4
, triclinic,
P
1̄ (no. 2),
a
= 9.9626(3) Å,
b
= 11.0489(3) Å,
c
= 12.3604(3) Å,
α
= 106.205(3)°,
β
= 99.165(2)°,
γ
= 100.306(3)°,
V
= 1253.53(6) Å
3
,
Z
= 1,
R
gt
...(
F
) = 0.0232,
wR
ref
(
F
2
) = 0.0555,
T
= 100(2) K.
The title trinuclear compound, Cu
(C
H
NS
)Cl
(C
H
P)
, has the di-thio-carbamate ligand symmetrically chelating one Cu
atom and each of the S atoms bridging to another Cu
atom. Both chloride ligands ...are bridging, one being μ
- and the other μ
-bridging. Each Et
P ligand occupies a terminal position. Two of the Cu
atoms exist within Cl
PS donor sets and the third is based on a ClPS
donor set, with each coordination geometry based on a distorted tetra-hedron. The constituents defining the core of the mol-ecule,
Cu
Cl
S
, occupy seven corners of a distorted cube. In the crystal, linear supra-molecular chains along the
axis are formed
phosphane-methyl-ene-C-H⋯Cl and pyrrolidine-methyl-ene-C-H⋯π(chelate) inter-actions, and these chains pack without directional inter-actions between them. An analysis of the Hirshfeld surface points to the predominance of H atoms at the surface,
contributing 86.6% to the surface, and also highlights the presence of C-H⋯π(chelate) inter-actions.
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