Sourajit M. Mustafi

Analysing the visible conformational substates of the FK506-binding protein FKBP12.

The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80′s loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the β-sheet, across the active site to the α-helix and into the 50′s loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80′s loop. Line-broadening was also decreased for the residues in the α-helix and 50′s loop, whereas line-broadening in the 40′s loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40′s loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.

Differential conformational dynamics in the closely homologous FK506-binding domains of FKBP51 and FKBP52

As co-chaperones of Hsp90 (heat-shock protein 90), FKBP51 (FK506-binding protein of 51 kDa) and FKBP52 (FK506-binding protein of 52 kDa) act as antagonists in regulating the hormone affinity and nuclear transport of steroid receptor complexes. Exchange of Leu119 in FKBP51 for Pro119 in FKBP52 has been shown to largely reverse the steroid receptor activities of FKBP51 and FKBP52. To examine whether differences in conformational dynamics/plasticity might correlate with changes in the reported receptor activities, 15N-NMR relaxation measurements were carried out on the N-terminal FKBP domains of FKBP51 and FKBP52 as well as their residue-swapped variants. Both proteins exhibit a similar pattern of motion in the picosecond–nanosecond timeframe as well as a small degree of 15N line-broadening, indicative of motion in the microsecond–millisecond timeframe, in the β3a strand of the central sheet. Only the FKBP51 domain exhibits much larger line-broadening in the adjacent β3 bulge (40′s loop of FKBP12) and throughout the long β4–β5 loop (80′s loop of FKBP12). The L119P mutation at the tip of the β4–β5 loop completely suppressed the line-broadening in this loop while partially suppressing the line-broadening in the neighbouring β2 and β3a strands. The complementary P119L and P119L/P124S variants of FKBP52 yielded similar patterns of line-broadening for the β4–β5 loop as that for FKBP51, although only 20% and 60% as intense respectively. However, despite the close structural similarity in the packing interactions between the β4–β5 loop and the β3a strand for FKBP51 and FKBP52, the line-broadening in the β3a strand is unaffected by the P119L or P119L/P124S mutations in FKBP52.

Coupling of Conformational Transitions in the N-terminal Domain of the 51-kDa FK506-binding Protein (FKBP51) Near Its Site of Interaction with the Steroid Receptor Proteins

Background: FK506-binding protein 51 (FKBP51) inhibits and FKBP52 stimulates transcription by various steroid receptors. Exchange of a single residue largely reverses this pattern of regulation. Results: Unlike FKBP52, FKBP51 has two distinct coupled conformational transitions surrounding this mutation site. Conclusion: Structural analysis of FKBP51 transient states can inform inhibitor design by conformational selection. Significance: The differential plasticity of the FKBP domains may underlie their differential regulation. Interchanging Leu-119 for Pro-119 at the tip of the β4-β5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the β4-β5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying β2 and β3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the β2 and β3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.

Structural basis of conformational transitions in the active site and 80's loop in the FK506-binding protein FKBP12.

The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80′s loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80′s loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans–cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80′s loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80′s loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50′s loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue.

Statistical allosteric coupling to the active site indole ring flip equilibria in the FK506-binding domain

In solution, the Trp 59 indole ring at the base of the active site cleft in the FKBP domain protein FKBP12 is rotated by ~90° at a population level of 20%, relative to its canonical crystallographic orientation. NMR measurements on the homologous FK1 domains of human FKBP51 and FKBP52 indicate no observable indole ring flip conformation, while the V101I variant of FKBP12 decreases the population having a perpendicular indole orientation by 10-fold. A set of three parallel 400 ns CHARMM27 molecular simulations for both wild type FKBP12 and the V101I variant examined how this ring flip might be energetically coupled to a transition of the Glu 60 sidechain which interacts with the backbone of the 50s loop located ~12 Å from the indole nitrogen. Analysis of the transition matrix for the local dynamics of the Glu 60 sidechain, the Trp 59 sidechain, and of the structurally interposed α-helix hydrogen bonding pattern yielded a statistical allosteric coupling of 10 kJ/mol with negligible concerted dynamical coupling for the transitions of the two sidechains.

Crystal structure and conformational flexibility of the unligated FK506-binding protein FKBP12.6.

Two crystal forms of unligated FKBP12.6 exhibit multiple conformations in the active site and in the 80s loop, the primary site for known protein-recognition interactions. The previously unreported NMR backbone assignment of FKBP12.6 revealed extensive doubling of amide resonances, which reflects a slow conformational transition centered in the 80s loop.

Acute White-Matter Abnormalities in Sports-Related Concussion: A Diffusion Tensor Imaging Study from the NCAA-DoD CARE Consortium

Sports-related concussion (SRC) is an important public health issue. Although standardized assessment tools are useful in the clinical management of acute concussion, the underlying pathophysiology of SRC and the time course of physiological recovery after injury remain unclear. In this study, we used diffusion tensor imaging (DTI) to detect white matter alterations in football players within 48 h after SRC. As part of the NCAA-DoD CARE Consortium study of SRC, 30 American football players diagnosed with acute concussion and 28 matched controls received clinical assessments and underwent advanced magnetic resonance imaging scans. To avoid selection bias and partial volume effects, whole-brain skeletonized white matter was examined by tract-based spatial statistics to investigate between-group differences in DTI metrics and their associations with clinical outcome measures. Mean diffusivity was significantly higher in brain white matter of concussed athletes, particularly in frontal and subfrontal long white matter tracts. In the concussed group, axial diffusivity was significantly correlated with the Brief Symptom Inventory and there was a similar trend with the symptom severity score of the Sport Concussion Assessment Tool. In addition, concussed athletes with higher fractional anisotropy performed better on the cognitive component of the Standardized Assessment of Concussion. Overall, the results of this study are consistent with the hypothesis that SRC is associated with changes in white matter tracts shortly after injury, and these differences are correlated clinically with acute symptoms and functional impairments.

Hybrid Diffusion Imaging in Mild Traumatic Brain Injury

Abstract Mild traumatic brain injury (mTBI) is an important public health problem. Although conventional medical imaging techniques can detect moderate-to-severe injuries, they are relatively insensitive to mTBI. In this study, we used hybrid diffusion imaging (HYDI) to detect white matter alterations in 19 patients with mTBI and 23 other trauma control patients. Within 15 days (standard deviation = 10) of brain injury, all subjects underwent magnetic resonance HYDI and were assessed with a battery of neuropsychological tests of sustained attention, memory, and executive function. Tract-based spatial statistics (TBSS) was used for voxel-wise statistical analyses within the white matter skeleton to study between-group differences in diffusion metrics, within-group correlations between diffusion metrics and clinical outcomes, and between-group interaction effects. The advanced diffusion imaging techniques, including neurite orientation dispersion and density imaging (NODDI) and q-space analyses, appeared to be more sensitive then classic diffusion tensor imaging. Only NODDI-derived intra-axonal volume fraction (Vic) demonstrated significant group differences (i.e., 5–9% lower in the injured brain). Within the mTBI group, Vic and a q-space measure, P0, correlated with 6 of 10 neuropsychological tests, including measures of attention, memory, and executive function. In addition, the direction of correlations differed significantly between groups (R2 > 0.71 and pinteration < 0.03). Specifically, in the control group, higher Vic and P0 were associated with better performances on clinical assessments, whereas in the mTBI group, higher Vic and P0 were associated with worse performances with correlation coefficients >0.83. In summary, the NODDI-derived axonal density index and q-space measure for tissue restriction demonstrated superior sensitivity to white matter changes shortly after mTBI. These techniques hold promise as a neuroimaging biomarker for mTBI.

ALTERATIONS IN WHITE-MATTER DIFFUSION METRICS IN PRECLINICAL ALZHEIMER’S DISEASE: A SUBJECT-SPECIFIC ANALYSIS

igure 2. Anatomical locations of extreme voxels for the diffusion metrics six SCD subjects labeled by S1 to S6. Nahla M. H. Elsaid, Qiuting Wen, Sourajit Mitra Mustafi, Shannon L. Risacher, Martin R. Farlow, Liana G. Apostolova, Andrew J. Saykin, Jaroslaw Harezlak, Yu-Chien Wu, Indiana Alzheimer Disease Center, Indianapolis, IN, USA; Department of Radiology and Imaging Sciences, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana University School of Medicine, Indianapolis, IN, USA; Department of Neurology, Indiana University School of Medicine, Indianapolis, IN, USA; School of Public Health, Indiana University, Bloomington, IN, USA. Contact e-mail: nelsaid@iu.edu

WHITE-MATTER MICROSTRUCTURE IN EARLY STAGE ALZHEIMER’S DISEASE

P3-396 WHITE-MATTER MICROSTRUCTURE IN EARLY STAGE ALZHEIMER’S DISEASE Qiuting Wen, Sourajit Mitra Mustafi, Jaroslaw Harezlak, Junjie Li, Shannon L. Risacher, John D. West, Eileen F. Tallman, Martin R. Farlow, Fred W. Unverzagt, Liana G. Apostolova, Andrew J. Saykin, Yu-Chien Wu, Indiana University School of Medicine, Indianapolis, IN, USA; Indiana Alzheimer Disease Center, Indianapolis, IN, USA; School of Public Health, Indiana University, Bloomington, IN, USA; Indiana University, Indianapolis, IN, USA. Contact e-mail: wenq@iu.edu