J. Patrick Loria

Characterization of Enzyme Motions by Solution NMR Relaxation Dispersion

In many enzymes, conformational changes that occur along the reaction coordinate can pose a bottleneck to the rate of conversion of substrates to products. Characterization of these rate-limiting protein motions is essential for obtaining a full understanding of enzyme-catalyzed reactions. Solution NMR experiments such as the Carr-Purcell-Meiboom-Gill (CPMG) spin-echo or off-resonance R 1rho pulse sequences enable quantitation of protein motions in the time range of microseconds to milliseconds. These experiments allow characterization of the conformational exchange rate constant, k ex, the equilibrium populations of the relevant conformations, and the chemical shift differences (Deltaomega) between the conformations. The CPMG experiments were applied to the backbone N-H positions of ribonuclease A (RNase A). To probe the role of dynamic processes in the catalytic cycle of RNase A, stable mimics of the apo enzyme (E), enzyme-substrate (ES) complex, and enzyme-product (EP) complex were formed. The results indicate that the ligand has relatively little influence on the kinetics of motion, which occurs at 1700 s (-1) and is the same as both k cat, and the product dissociation rate constant. Instead, the effect of ligand is to stabilize one of the pre-existing conformations. Thus, these NMR experiments indicate that the conformational change in RNase A is ligand-stabilized and does not appear to be ligand-induced. Further evidence for the coupling of motion and enzyme function comes from the similar solvent deuterium kinetic isotope effect on k ex derived from the NMR measurements and k cat from enzyme kinetic studies. This isotope effect of approximately 2 depends linearly on solvent deuterium content suggesting the involvement of a single proton in RNase A motion and function. Moreover, mutation of His48 to alanine eliminates motion in RNase A and decreases the catalytic turnover rate indicating the involvement of His48, which is far from the active site, in coupling motion and function. For the enzyme triosephosphate isomerase (TIM), the opening and closing motion of a highly conserved active site loop (loop 6) has been implicated in many studies to play an important role in the catalytic cycle of the enzyme. Off-resonance R 1rho experiments were performed on TIM, and results were obtained for amino acid residues in the N-terminal (Val167), and C-terminal (Lys174, Thr177) portions of loop 6. The results indicate that all three loop residues move between the open and closed conformation at about 10,000 s (-1), which is the same as the catalytic rate constant. The O (eta) atom of Tyr208 provides a hydrogen bond to stabilize the closed form of loop 6 by interacting with the amide nitrogen of Ala176; these atoms are outside of hydrogen bonding distance in the open form of the enzyme. Mutation of Tyr208 to phenylalanine results in significant loss of catalytic activity but does not appear to alter the kex value of the N-terminal part of loop 6. Instead, removal of this hydrogen bond appears to result in an increase in the equilibrium population of the open conformer of loop 6, thereby resulting in a loss of activity through a shift in the conformational equilibrium of loop 6. Solution NMR relaxation dispersion experiments are powerful experimental tools that can elucidate protein motions with atomic resolution and can provide insight into the role of these motions in biological function.

Helix stabilization precedes aqueous and bilayer catalyzed fiber formation in islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-beta-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples alpha-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate beta-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.

The mechanism of rate-limiting motions in enzyme function

The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of ≈160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.

Allosteric pathways in imidazole glycerol phosphate synthase

Protein allosteric pathways are investigated in the imidazole glycerol phosphate synthase heterodimer in an effort to elucidate how the effector (PRFAR, N′-[(5′-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) activates glutaminase catalysis at a distance of 25 Å from the glutamine-binding site. We apply solution NMR techniques and community analysis of dynamical networks, based on mutual information of correlated protein motions in the active and inactive enzymes. We find evidence that the allosteric pathways in the PRFAR bound enzyme involve conserved residues that correlate motion of the PRFAR binding loop to motion at the protein-protein interface, and ultimately at the glutaminase active site. The imidazole glycerol phosphate synthase bienzyme is an important branch point for the histidine and nucleotide biosynthetic pathways and represents a potential therapeutic target against microbes. The proposed allosteric mechanism and the underlying allosteric pathways provide fundamental insights for the design of new allosteric drugs and/or alternative herbicides.

Conformational Motions Regulate Phosphoryl Transfer in Related Protein Tyrosine Phosphatases

Closing the Loop Many studies have shown that protein dynamics are important to enzyme function. For example, enzyme protein movements have been shown to optimize the active site, enable binding of substrate and cofactor, and facilitate product release. Whittier et al. (p. 899) now show that in two tyrosine phosphatases, the rate of cleavage is coupled to motion of a loop. The two phosphatases have different catalytic rates; however, in both, a loop containing a catalytic residue switches between an inactive open and a catalytically competent closed state. The rates of closure are equivalent to the cleavage rates, suggesting that the leaving group tyrosine is protonated simultaneously with loop closure. Thus, tuning of the loop motion plays a regulatory role in the catalytic cycle. Differences in the rate of an internal protein loop closure are coupled to differences in enzyme reaction rates. Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using nuclear magnetic resonance spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates; however, we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes.

NMR insights into protein allostery

Allosterism is one of natures principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.

Protein dynamics from solution NMR

Solution nuclear magnetic resonance (NMR) spectroscopy is unique in its ability to elucidate the details of atomic-level structural and dynamical properties of biological macromolecules under native-like conditions. Recent advances in NMR techniques and protein sample preparation now allow comprehensive investigation of protein dynamics over timescales ranging 14 orders of magnitude at nearly every atomic site. Thus, solution NMR is poised to reveal aspects of the physico-chemical properties that govern the ensemble distribution of protein conformers and the dynamics of their interconversion. We review these advances as well as their recent application to the study of proteins.

Role of loop-loop interactions in coordinating motions and enzymatic function in triosephosphate isomerase.

The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homologue caused a 10(2)-fold loss in enzymatic activity, a decrease in substrate binding affinity, and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional (1)H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R(1rho) experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and that the coordinated motion of loop 6 is reduced relative to that of the WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur and that the interactions play a significant role in modulating the chemical dynamics near the active site.

The effects of cosolutes on protein dynamics: The reversal of denaturant-induced protein fluctuations by trimethylamine N-oxide

The protein stabilizing effects of the small molecule osmolyte, trimethylamine N‐oxide, against chemical denaturant was investigated by NMR spin‐relaxation measurements and model‐free analysis. In the presence of 0.7 M guanidine hydrochloride increased picosecond‐nanosecond dynamics are observed in the protein ribonuclease A. These increased fluctuations occur throughout the protein, but the most significant increases in flexibility occur at positions believed to be the first to unfold. Addition of 0.35 M trimethylamine N‐oxide to this destabilized form of ribonuclease results in significant rigidification of the protein backbone as assessed by 1H‐15N order parameters. Statistically, these order parameters are the same as those measured in native ribonuclease indicating that TMAO reduces the amplitude of backbone fluctuations in a destabilized protein. These data suggest that TMAO restricts the bond vector motions on the protein energy landscape to resemble those motions that occur in the native protein and points to a relation between stability and dynamics in this enzyme.

Solution NMR and computational methods for understanding protein allostery.

Allosterism is an essential biological regulatory mechanism. In enzymes, allosteric regulation results in an activation or inhibition of catalytic turnover. The mechanisms by which this is accomplished are unclear and vary significantly depending on the enzyme. It is commonly the case that a metabolite binds to the enzyme at a site distant from the catalytic site, yet its binding is coupled to and sensed by the active site. This coupling can manifest in changes in structure, dynamics, or both at the active site. These interactions between the allosteric and active site, which are often quite distant from one another, involve numerous atoms as well as complex conformational rearrangements of the protein secondary and tertiary structure. Interrogation of this complex biological phenomenon necessitates multiple experimental approaches. In this article, we outline a combined solution NMR spectroscopic and computational approach using molecular dynamics and network models to uncover mechanistic aspects of allostery in the enzyme imidazole glycerol phosphate synthase.