Evaluating protein cross-linking as a therapeutic strategy to stabilize SOD1 variants in a mouse model of familial ALS.

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.

A Model for the Solution Structure of Human Fe(II)-Bound Acireductone Dioxygenase and Interactions with the Regulatory Domain of Matrix Metalloproteinase I (MMP-I).

The metalloenzyme acireductone dioxygenase (ARD) shows metal-dependent physical and enzymatic activities depending upon the metal bound in the active site. The Fe(II)-bound enzyme catalyzes the penultimate step of the methionine salvage pathway, converting 1,2-dihydroxy-5-(methylthio)pent-1-en-3-one (acireductone) into formate and the ketoacid precursor of methionine, 2-keto-4-thiomethyl-2-oxobutanoate, using O2 as the oxidant. If Ni(II) is bound, an off-pathway shunt occurs, producing 3-methylthiopropionate, formate, and carbon monoxide from the same acireductone substrate. The solution structure of the Fe(II)-bound human enzyme, HsARD, is described and compared with the structures of Ni-bound forms of the closely related mouse enzyme, MmARD. Potential rationales for the different reactivities of the two isoforms are discussed. The human enzyme has been found to regulate the activity of matrix metalloproteinase I (MMP-I), which is involved in tumor metastasis, by binding the cytoplasmic transmembrane tail peptide of MMP-I. Nuclear magnetic resonance titration of HsARD with the MMP-I tail peptide permits identification of the peptide binding site on HsARD, a cleft anterior to the metal binding site adjacent to a dynamic proline-rich loop.

Crystal Structures of Cystathionine ß-Synthase from Saccharomyces cerevisiae: One Enzymatic Step at a Time.

Cystathionine ß-synthase (CBS) is a key regulator of sulfur amino acid metabolism, taking homocysteine from the methionine cycle to the biosynthesis of cysteine via the trans-sulfuration pathway. CBS is also a predominant source of H2S biogenesis. Roles for CBS have been reported for neuronal death pursuant to cerebral ischemia, promoting ovarian tumor growth, and maintaining drug-resistant phenotype by controlling redox behavior and regulating mitochondrial bioenergetics. The trans-sulfuration pathway is well-conserved in eukaryotes, but the analogous enzymes have different enzymatic behavior in different organisms. CBSs from the higher organisms contain a heme in an N-terminal domain. Though the presence of the heme, whose functions in CBSs have yet to be elucidated, is biochemically interesting, it hampers UV-vis absorption spectroscopy investigations of pyridoxal 5'-phosphate (PLP) species. CBS from Saccharomyces cerevisiae (yCBS) naturally lacks the heme-containing N-terminal domain, which makes it an ideal model for spectroscopic studies of the enzymological reaction catalyzed and allows structural studies of the basic yCBS catalytic core (yCBS-cc). Here we present the crystal structure of yCBS-cc, solved to 1.5 Å. Crystal structures of yCBS-cc in complex with enzymatic reaction intermediates have been captured, providing a structural basis for residues involved in catalysis. Finally, the structure of the yCBS-cc cofactor complex generated by incubation with an inhibitor shows apparent off-pathway chemistry not normally seen with CBS.

Selective Targeting by a Mechanism-Based Inactivator against Pyridoxal 5'-Phosphate-Dependent Enzymes: Mechanisms of Inactivation and Alternative Turnover.

Potent mechanism-based inactivators can be rationally designed against pyridoxal 5'-phosphate (PLP)-dependent drug targets, such as ornithine aminotransferase (OAT) or γ-aminobutyric acid aminotransferase (GABA-AT). An important challenge, however, is the lack of selectivity toward other PLP-dependent, off-target enzymes, because of similarities in mechanisms of all PLP-dependent aminotransferase reactions. On the basis of complex crystal structures, we investigate the inactivation mechanism of OAT, a hepatocellular carcinoma target, by (1R,3S,4S)-3-amino-4-fluorocyclopentane-1-carboxylic acid (FCP), a known inactivator of GABA-AT. A crystal structure of OAT and FCP showed the formation of a ternary adduct. This adduct can be rationalized as occurring via an enamine mechanism of inactivation, similar to that reported for GABA-AT. However, the crystal structure of an off-target, PLP-dependent enzyme, aspartate aminotransferase (Asp-AT), in complex with FCP, along with the results of attempted inhibition assays, suggests that FCP is not an inactivator of Asp-AT, but rather an alternate substrate. Turnover of FCP by Asp-AT is also supported by high-resolution mass spectrometry. Amid existing difficulties in achieving selectivity of inactivation among a large number of PLP-dependent enzymes, the obtained results provide evidence that a desirable selectivity could be achieved, taking advantage of subtle structural and mechanistic differences between a drug-target enzyme and an off-target enzyme, despite their largely similar substrate binding sites and catalytic mechanisms.

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase.

Acireductone dioxygenase (ARD) from the methionine salvage pathway (MSP) is a unique enzyme that exhibits dual chemistry determined solely by the identity of the divalent transition-metal ion (Fe2+ or Ni2+) in the active site. The Fe2+-containing isozyme catalyzes the on-pathway reaction using substrates 1,2-dihydroxy-3-keto-5-methylthiopent-1-ene (acireductone) and dioxygen to generate formate and the ketoacid precursor of methionine, 2-keto-4-methylthiobutyrate, whereas the Ni2+-containing isozyme catalyzes an off-pathway shunt with the same substrates, generating methylthiopropionate, carbon monoxide, and formate. The dual chemistry of ARD was originally discovered in the bacterium Klebsiella oxytoca, but it has recently been shown that mammalian ARD enzymes (mouse and human) are also capable of catalyzing metal-dependent dual chemistry in vitro. This is particularly interesting, since carbon monoxide, one of the products of off-pathway reaction, has been identified as an antiapoptotic molecule in mammals. In addition, several biochemical and genetic studies have indicated an inhibitory role of human ARD in cancer. This comprehensive review describes the biochemical and structural characterization of the ARD family, the proposed experimental and theoretical approaches to establishing mechanisms for the dual chemistry, insights into the mechanism based on comparison with structurally and functionally similar enzymes, and the applications of this research to the field of artificial metalloenzymes and synthetic biology.

Structure and mechanism of benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633, a member of the Class 3 aldehyde dehydrogenase superfamily.

Benzaldehyde dehydrogenase from Pseudomonas putida (PpBADH) belongs to the Class 3 aldehyde dehydrogenase (ALDH) family. The Class 3 ALDHs are unusual in that they are generally dimeric (rather than tetrameric), relatively non-specific and utilize both NAD+ and NADP+. To date, X-ray structures of three Class 3 ALDHs have been determined, of which only two have cofactor bound, both in the NAD+ form. Here we report the crystal structure of PpBADH in complex with NADP+ and a thioacyl intermediate adduct. The overall architecture of PpBADH resembles that of most other members of the ALDH superfamily, and the cofactor binding residues are well conserved. Conversely, the pattern of cofactor binding for the rat Class 3 ALDH differs from that of PpBADH and other ALDHs. This has been interpreted in terms of a different mechanism for the rat enzyme. Comparison with the PpBADH structure, as well as multiple sequence alignments, suggest that one of two conserved glutamates, at positions 215 (209 in rat) and 337 (333 in rat), would act as the general base necessary to hydrolyze the thioacyl intermediate. While the latter is the general base in the rat Class 3 ALDH, site-specific mutagenesis indicates that Glu215 is the likely candidate for PpBADH, a result more typical of the Class 1 and 2 ALDH families. Finally, this study shows that hydride transfer is not rate limiting, lending further credence to the suggestion that PpBADH is more similar to the Class 1 and 2 ALDHs than it is to other Class 3 ALDHs.

PLP and GABA trigger GabR-mediated transcription regulation in Bacillus subtilis via external aldimine formation.

The Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c).

The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.

Dual chemistry catalyzed by human acireductone dioxygenase.

Acireductone dioxygenase (ARD) from the methionine salvage pathway of Klebsiella oxytoca is the only known naturally occurring metalloenzyme that catalyzes different reactions in vivo based solely on the identity of the divalent transition metal ion (Fe2+ or Ni2+) bound in the active site. The iron-containing isozyme catalyzes the cleavage of substrate 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, whereas the nickel-containing isozyme uses the same substrates to catalyze an off-pathway shunt to form methylthiopropionate, carbon monoxide and formate. This dual chemistry was recently demonstrated in vitro by ARD from Mus musculus (MmARD), providing the first example of a mammalian ARD exhibiting metal-dependent catalysis. We now show that human ARD (HsARD) is also capable of metal-dependent dual chemistry. Recombinant HsARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. As with MmARD, the Fe2+-bound HsARD shows the highest activity and catalyzes on-pathway chemistry, whereas Ni2+, Co2+ or Mn2+ forms catalyze off-pathway chemistry. The thermal stability of the HsARD isozymes is a function of the metal ion identity, with Ni2+-bound HsARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound HsARD being the least stable. As with the bacterial ARD, solution NMR data suggest that HsARD isozymes can have significant structural differences depending upon the metal ion bound.

Crystal structure of the DNA binding domain of the transcription factor T-bet suggests simultaneous recognition of distant genome sites.

The transcription factor T-bet (Tbox protein expressed in T cells) is one of the master regulators of both the innate and adaptive immune responses. It plays a central role in T-cell lineage commitment, where it controls the TH1 response, and in gene regulation in plasma B-cells and dendritic cells. T-bet is a member of the Tbox family of transcription factors; however, T-bet coordinately regulates the expression of many more genes than other Tbox proteins. A central unresolved question is how T-bet is able to simultaneously recognize distant Tbox binding sites, which may be located thousands of base pairs away. We have determined the crystal structure of the Tbox DNA binding domain (DBD) of T-bet in complex with a palindromic DNA. The structure shows a quaternary structure in which the T-bet dimer has its DNA binding regions splayed far apart, making it impossible for a single dimer to bind both sites of the DNA palindrome. In contrast to most other Tbox proteins, a single T-bet DBD dimer binds simultaneously to identical half-sites on two independent DNA. A fluorescence-based assay confirms that T-bet dimers are able to bring two independent DNA molecules into close juxtaposition. Furthermore, chromosome conformation capture assays confirm that T-bet functions in the direct formation of chromatin loops in vitro and in vivo. The data are consistent with a looping/synapsing model for transcriptional regulation by T-bet in which a single dimer of the transcription factor can recognize and coalesce distinct genetic elements, either a promoter plus a distant regulatory element, or promoters on two different genes.

Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

Reducing C-terminal truncation mitigates synucleinopathy and neurodegeneration in a transgenic model of multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic orphan neurodegenerative disorder. No treatment is currently available to slow down the aggressive neurodegenerative process, and patients die within a few years after disease onset. The cytopathological hallmark of MSA is the accumulation of alpha-synuclein (α-syn) aggregates in affected oligodendrocytes. Several studies point to α-syn oligomerization and aggregation as a mediator of neurotoxicity in synucleinopathies including MSA. C-terminal truncation by the inflammatory protease caspase-1 has recently been implicated in the mechanisms that promote aggregation of α-syn in vitro and in neuronal cell models of α-syn toxicity. We present here an in vivo proof of concept of the ability of the caspase-1 inhibitor prodrug VX-765 to mitigate α-syn pathology and to mediate neuroprotection in proteolipid protein α-syn (PLP-SYN) mice, a transgenic mouse model of MSA. PLP-SYN and age-matched wild-type mice were treated for a period of 11 wk with VX-765 or placebo. VX-765 prevented motor deficits in PLP-SYN mice compared with placebo controls. More importantly, VX-765 was able to limit the progressive toxicity of α-syn aggregation by reducing its load in the striatum of PLP-SYN mice. Not only did VX-765 reduce truncated α-syn, but it also decreased its monomeric and oligomeric forms. Finally, VX-765 showed neuroprotective effects by preserving tyrosine hydroxylase-positive neurons in the substantia nigra of PLP-SYN mice. In conclusion, our results suggest that VX-765, a drug that was well tolerated in a 6 wk-long phase II trial in patients with epilepsy, is a promising candidate to achieve disease modification in synucleinopathies by limiting α-syn accumulation.

Metal-Dependent Function of a Mammalian Acireductone Dioxygenase.

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe(2+) or Ni(2+)) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate the metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, which is the penultimate step in methionine salvage. The nickel-bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO), and 3-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe(2+)-bound protein, which shows about 10-fold higher activity than that of others, catalyzes on-pathway chemistry, whereas the Ni(2+), Co(2+), or Mn(2+) forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by the metal ion identity, with Ni(2+)-bound MmARD being the most stable, followed by Co(2+) and Fe(2+), and Mn(2+)-bound ARD being the least stable. Ni(2+)- and Co(2+)-bound MmARD were crystallized, and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination, and the catalytic mechanism.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson's disease.

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1.

Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNA-binding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43- and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.

Prediction of distal residue participation in enzyme catalysis.

A scoring method for the prediction of catalytically important residues in enzyme structures is presented and used to examine the participation of distal residues in enzyme catalysis. Scores are based on the Partial Order Optimum Likelihood (POOL) machine learning method, using computed electrostatic properties, surface geometric features, and information obtained from the phylogenetic tree as input features. Predictions of distal residue participation in catalysis are compared with experimental kinetics data from the literature on variants of the featured enzymes; some additional kinetics measurements are reported for variants of Pseudomonas putida nitrile hydratase (ppNH) and for Escherichia coli alkaline phosphatase (AP). The multilayer active sites of P. putida nitrile hydratase and of human phosphoglucose isomerase are predicted by the POOL log ZP scores, as is the single-layer active site of P. putida ketosteroid isomerase. The log ZP score cutoff utilized here results in over-prediction of distal residue involvement in E. coli alkaline phosphatase. While fewer experimental data points are available for P. putida mandelate racemase and for human carbonic anhydrase II, the POOL log ZP scores properly predict the previously reported participation of distal residues.

The use of pharmacological retromer chaperones in Alzheimer's disease and other endosomal-related disorders.

The retromer is an evolutionary conserved multiprotein complex involved in the sorting and retrograde trafficking of cargo from endosomal compartments to the Golgi network and to the cell surface. The neuronal retromer traffics the amyloid precursor protein away from the endosomes, a site where amyloid precursor protein is enzymatically cleaved into pathogenic fragments in Alzheimer's disease. In recent years, deficiencies in retromer-mediated transport have been implicated in several neurological and non-neurological diseases, including Parkinson's disease, suggesting that improving the efficacy of the retromer trafficking pathway would result in decreased pathology. We recently identified a new family of small molecules that appear to stabilize the interaction between members of the retromer complex and enhance its function in neurons: the retromer pharmacological chaperones. Here we discuss the role of these molecules in the improvement of retromer trafficking and endosomal dysfunction, as well as their potential as therapeutics for neurological and non-neurological disorders.

Crystal cryocooling distorts conformational heterogeneity in a model Michaelis complex of DHFR.

Most macromolecular X-ray structures are determined from cryocooled crystals, but it is unclear whether cryocooling distorts functionally relevant flexibility. Here we compare independently acquired pairs of high-resolution data sets of a model Michaelis complex of dihydrofolate reductase (DHFR), collected by separate groups at both room and cryogenic temperatures. These data sets allow us to isolate the differences between experimental procedures and between temperatures. Our analyses of multiconformer models and time-averaged ensembles suggest that cryocooling suppresses and otherwise modifies side-chain and main-chain conformational heterogeneity, quenching dynamic contact networks. Despite some idiosyncratic differences, most changes from room temperature to cryogenic temperature are conserved and likely reflect temperature-dependent solvent remodeling. Both cryogenic data sets point to additional conformations not evident in the corresponding room temperature data sets, suggesting that cryocooling does not merely trap preexisting conformational heterogeneity. Our results demonstrate that crystal cryocooling consistently distorts the energy landscape of DHFR, a paragon for understanding functional protein dynamics.