Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures

Holliday, Michael J.; Camilloni, Carlo; Armstrong, Geoffrey S.; Isern, Nancy G.; Zhang, Fengli; Vendruscolo, Michele; Eisenmesser, Elan

doi:10.1021/acs.biochem.5b00263

Cited by 19 publications

(54 citation statements)

References 78 publications

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“…33 We have previously demonstrated that CypA and GeoCyp both bind to and catalyze isomerization of this peptide. 24 Here, we have also characterized the activities of two additional human cyclophilins, CypB and CypC, toward this substrate. The chemical shifts of CypA, 25 CypB, 27 and GeoCyp 26 have been previously published, but not those for CypC.…”

Section: Resultsmentioning

confidence: 99%

“…24 Multiple residues exhibit distinct cis and trans amide chemical shifts, including two resides (Asp 6 and Leu 7) for which the cis, trans , and cross-peaks are resolvable (one cross-peak for Asp 6 is overlapped with another residue peak). The time-dependent disappearance of the cis and trans peaks and appearance of cross-peaks can be used to determine an isomerization rate ( k iso ) between the two free isoforms under the given conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Titration data were collected at 10 °C on a Varian 900 MHz spectrometer with a cryogenically cooled probe and fit as previously described. 24 …”

Section: Methodsmentioning

confidence: 99%

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Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

2015

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Cyclophilins catalyze cis ↔ trans isomerization of peptidyl–prolyl bonds, influencing protein folding along with a breadth of other biological functions such as signal transduction. Here, we have determined the microscopic rate constants defining the full enzymatic cycle for three human cyclophilins and a more distantly related thermophilic bacterial cyclophilin when catalyzing interconversion of a biologically representative peptide substrate. The cyclophilins studied here exhibit variability in on-enzyme interconversion as well as an up to 2-fold range in rates of substrate binding and release. However, among the human cyclophilins, the microscopic rate constants appear to have been tuned to maintain remarkably similar isomerization rates without a concurrent conservation of apparent binding affinities. While the structures and active site compositions of the human cyclophilins studied here are highly conserved, we find that the enzymes exhibit significant variability in microsecond to millisecond time scale mobility, suggesting a role for the inherent conformational fluctuations that exist within the cyclophilin family as being functionally relevant in regulating substrate interactions. We have additionally modeled the relaxation dispersion profile given by the commonly employed Carr–Purcell–Meiboom–Gill relaxation dispersion (CPMG-RD) experiment when applied to a reversible enzymatic system such as cyclophilin isomerization and identified a significant limitation in the applicability of this approach for monitoring on-enzyme turnover. Specifically, we show both computationally and experimentally that the CPMG-RD experiment is sensitive to noncatalyzed substrate binding and release in reversible systems even at saturating substrate concentrations unless the on-enzyme interconversion rate is much faster than the substrate release rate.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

2015

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“…The NMR chemical shifts were determined as previously described (41). The catalytic isomerization rates were determined by ZZ exchange as previously described (41).…”

Section: Methodsmentioning

confidence: 99%

“…The NMR chemical shifts were determined as previously described (41). The catalytic isomerization rates were determined by ZZ exchange as previously described (41). More specifically, ZZ-exchange data were collected on 1 mM 15 N-labeled peptide with 20 μM unlabeled wild-type CypA or V29L CypA mutant at 10°C on a Varian 600 MHz spectrometer with a cryogenically cooled probe.…”

Section: Methodsmentioning

confidence: 99%

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Doshi

Holliday

Eisenmesser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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148

186

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Detailed understanding of how conformational dynamics orchestrates function in allosteric regulation of recognition and catalysis remains ambiguous. Here, we simulate CypA using multiple-microsecond-long atomistic molecular dynamics in explicit solvent and carry out NMR experiments. We analyze a large amount of time-dependent multidimensional data with a coarse-grained approach and map key dynamical features within individual macrostates by defining dynamics in terms of residue-residue contacts. The effects of substrate binding are observed to be largely sensed at a location over 15 Å from the active site, implying its importance in allostery. Using NMR experiments, we confirm that a dynamic cluster of residues in this distal region is directly coupled to the active site. Furthermore, the dynamical network of interresidue contacts is found to be coupled and temporally dispersed, ranging over 4 to 5 orders of magnitude. Finally, using network centrality measures we demonstrate the changes in the communication network, connectivity, and influence of CypA residues upon substrate binding, mutation, and during catalysis. We identify key residues that potentially act as a bottleneck in the communication flow through the distinct regions in CypA and, therefore, as targets for future mutational studies. Mapping these dynamical features and the coupling of dynamics to function has crucial ramifications in understanding allosteric regulation in enzymes and proteins, in general.allostery | enzyme dynamics | residue-residue contacts | Cyclophilin A | molecular dynamics

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Streptococcus pneumoniae G5 domains bind different ligands

et al. 2019

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Many bacterial pathogens express small G5 domains that exist in the context of various membrane‐anchored proteins and these G5 domains have been associated with colonization, cellular adhesion, and biofilm formation. However, despite over a decade since the computational prediction of these G5 domains, many remain uncharacterized, particularly those from Streptococcus pneumoniae. Of five previously predicted G5 domains we found that four of these, all derived from S. pneumoniae, are independently folded modules. As one of these exhibits extreme line broadening due to self‐association, we were able to use NMR solution studies to probe the potential ligand interactions of the remaining three G5 domains. None of these G5 domains engage N‐acetylglucosamine (NAG) as previously predicted but do interact with other small molecules that may modulate adherence to both bacteria and host cells. Specifically, while all G5 domains tested engage Zn, only one of these G5 domains engage heparin. NMR solution structural studies of the IgA1 Protease G5 (IgA1P‐G5) and endo‐beta‐N‐acetylglucosaminidase‐D G5 (ENDD‐G5) also facilitated identification of the ligand binding sites and confirm the typical G5 fold that comprises two connected β‐sheets with no canonical core. NMR relaxation experiments indicate flexibility on both ends and within the connecting regions between the β‐sheets. Our studies thus establish a basis for future biological experiments to test whether the ligands presented here are involved in bacterial adherence, either to bacteria or to host cells.

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Structure and Dynamics of GeoCyp: A Thermophilic Cyclophilin with a Novel Substrate Binding Mechanism That Functions Efficiently at Low Temperatures

Cited by 19 publications

References 78 publications

Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

Determination of the Full Catalytic Cycle among Multiple Cyclophilin Family Members and Limitations on the Application of CPMG-RD in Reversible Catalytic Systems

Dynamical network of residue–residue contacts reveals coupled allosteric effects in recognition, catalysis, and mutation

Streptococcus pneumoniae G5 domains bind different ligands

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