Dynamic Asymmetry and the Role of the Conserved Active-Site Thiol in Rabbit Muscle Creatine Kinase

Abstract

Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

Probing the effectiveness of spectroscopic reporter unnatural amino acids: a structural study

“…Unnatural amino acids (UAAs) have the potential to provide site-specific information about protein structure and dynamics (Getahun et al, 2003;Tucker et al, 2005;Schultz et al, 2006;Aprilakis et al, 2007;Jackson et al, 2007;Glasscock et al, 2008;Oh et al, 2008;Weeks et al, 2008;Miyake-Stoner et al, 2009;Taskent-Sezgin et al, 2009Waegele et al, 2009Waegele et al, , 2011Ye et al, 2009Ye et al, , 2010Fafarman & Boxer, 2010;Goldberg et al, 2010;Urbanek et al, 2010;Nagarajan et al, 2011;Thielges et al, 2011;Bazewicz et al, 2012Bazewicz et al, , 2013Peran et al, 2014;Petersson et al, 2014;Walker et al, 2014;Londergan et al, 2015;Tookmanian et al, 2015). Fundamentally, these spectroscopic reporter UAAs must contain a readily measurable spectroscopic signature that is sensitive to the local environment and must be able to be site-specifically incorporated into peptides or proteins with high efficiency and fidelity while being minimally intrusive.…”

Azidoethoxyphenylalanine as a Vibrational Reporter and Click Chemistry Partner in Proteins

“…[6] In the case of reporters, au nique spectroscopic signature that can be observed by fluorescence, [7][8][9][10] EPR, [11,12] NMR, [13,14] or vibrational spectroscopy [15][16][17] is required. In recent years vibrational UAA have been employed to study the local hydra-tion and the electrostatic environments of proteins by IR [18][19][20][21][22][23][24][25][26] and Raman spectroscopy. [26,27] Nitrilesa nd azides are arguably the two most important functional groups for vibrationalU AA becauseo ft heir small size, relative stability,s ensitivity,a nd absorptions that occur in an open window in the IR.…”

“…In recent years vibrational UAA have been employed to study the local hydra-tion and the electrostatic environments of proteins by IR [18][19][20][21][22][23][24][25][26] and Raman spectroscopy. [26,27] Nitrilesa nd azides are arguably the two most important functional groups for vibrationalU AA becauseo ft heir small size, relative stability,s ensitivity,a nd absorptions that occur in an open window in the IR. [18,28] We recentlyr eported ad irect comparison of these two vibrational reportersa nd demonstrated that for many applicationsa zides are superior because they have as ignificantly larger extinction coefficient while maintaining excellent sensitivity to their local environment in terms of frequency shifts.…”

The Monoclonal Antibody Recognized Apoptosis-Associated Protein in PCV2-Infected Peripheral Blood Mononuclear Cells

“…Intriguingly, each site contains cysteine residues. It is implied the thiol group of cysteine that might play a role in the binding affinity [47][48][49] was generated by immunological methods and hybridoma screening which was selected specifically binding to the 35th to 66th amino acid residues of ORF3 protein. Therefore, further investigation of the amino acid sequence of the antigen-binding sites of mAb 7D3 would be interesting.…”