The activation of oncogenic C-terminal binding Protein (CtBP) transcriptional activity is coupled with NAD(H) binding and homo-oligomeric assembly, although the level of CtBP assembly and nucleotide binding affinity continues to be debated. Here, we apply biophysical techniques to address these fundamental issues for CtBP1 and CtBP2. Our ultracentrifugation results unambiguously demonstrate that CtBP assembles into tetramers in the presence of saturating NAD(+) or NADH with tetramer to dimer dissociation constants about 100 nm. Isothermal titration calorimetry measurements of NAD(H) binding to CtBP show dissociation constants between 30 and 500 nm, depending on the nucleotide and paralog. Given cellular levels of NAD(+), CtBP is likely to be fully saturated with NAD under physiological concentrations suggesting that CtBP is unable to act as a sensor for NADH levels.

C-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis, and metastasis. NADH-dependent CtBP activation has been implicated in multiple types of cancer and poor patient prognosis. Central to understanding activation of CtBP in oncogenesis is uncovering how NADH triggers protein assembly, what level of assembly occurs, and if oncogenic activity depends upon such assembly. Here, we present the cryoelectron microscopic structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is tetrameric. The physiological relevance of the observed tetramer was demonstrated in cell culture, showing that CtBP tetramer-destabilizing mutants are defective for cell migration, transcriptional repression of E-cadherin, and activation of TIAM1. Together with our cryoelectron microscopy studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.

C-terminal binding proteins (CtBPs) are cotranscriptional factors that play key roles in cell fate. We have previously shown that NAD(H) promotes the assembly of similar tetramers from either human CtBP1 and CtBP2 and that CtBP2 tetramer destabilizing mutants are defective for oncogenic activity. To assist structure-based design efforts for compounds that disrupt CtBP tetramerization, it is essential to understand how NAD(H) triggers tetramer assembly. Here, we investigate the moieties within NAD(H) that are responsible for triggering tetramer formation. Using multiangle light scattering (MALS), we show that ADP is able to promote tetramer formation of both CtBP1 and CtBP2, whereas AMP promotes tetramer assembly of CtBP1, but not CtBP2. Other NAD(H) moieties that lack the adenosine phosphate, including adenosine and those incorporating nicotinamide, all fail to promote tetramer assembly. Our crystal structures of CtBP1 with AMP reveal participation of the adenosine phosphate in the tetrameric interface, pinpointing its central role in NAD(H)-linked assembly. CtBP1 and CtBP2 have overlapping but unique roles, suggesting that a detailed understanding of their unique structural properties might have utility in the design of paralog-specific inhibitors. We investigated the different responses to AMP through a series of site-directed mutants at 13 positions. These mutations reveal a central role for a hinge segment, which we term the 120s hinge that connects the substrate with coenzyme-binding domains and influences nucleotide binding and tetramer assembly. Our results provide insight into suitable pockets to explore in structure-based drug design to interfere with cotranscriptional activity of CtBP in cancer.

Twinning is a crystal-growth anomaly in which protein monomers exist in different orientations but are related in a specific way, causing diffraction reflections to overlap. Twinning imposes additional symmetry on the data, often leading to the assignment of a higher symmetry space group. Specifically, in merohedral twinning, reflections from each monomer overlap and require a twin law to model unique structural data from overlapping reflections. Neglecting twinning in the crystallographic analysis of quasi-rotationally symmetric homo-oligomeric protein structures can mask the degree of structural non-identity between monomers. In particular, any deviations from perfect symmetry will be lost if higher than appropriate symmetry is applied during crystallographic analysis. Such cases warrant choosing between the highest symmetry space group possible or determining whether the monomers have distinguishable structural asymmetries and thus require a lower symmetry space group and a twin law. Using hexagonal cocrystals of HIV-1 protease, a C-2-symmetric homodimer whose symmetry is broken by bound ligand, it is shown that both assigning a lower symmetry space group and applying a twin law during refinement are critical to achieving a structural model that more accurately fits the electron density. By re-analyzing three recently published HIV-1 protease structures, improvements in nearly every crystallographic metric are demonstrated. Most importantly, a procedure is demonstrated where the inhibitor can be reliably modeled in a single orientation. This protocol may be applicable to many other homo-oligomers in the PDB.

C-terminal binding proteins (CtBP1 & 2) are co-transcriptional factors that have been implicated in progression of a broad range of cancers. CtBP possesses a D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domain that binds NAD(H) and provides an attractive target for small molecule intervention. We have previously demonstrated that NAD(H) triggers assembly of CtBP1 and CtBP2 into structurally very similar tetramers, strongly suggesting that this tetrameric form is the co-transcriptionally active species. Using multi-angle light scattering (MALS) to test various NAD(H) moieties, we find that AMP and ADP, but not adenosine, nicotinamide and nicotinamide mononucleotide promote tetramer formation, demonstrating that the adenosine phosphate plays a central role in tetrameric assembly. Crystal structures with bound AMP show a direct structural role of this phosphate in stabilizing a key interaction across the tetrameric interface. Most importantly, these results indicate that development of inhibitors that extend into the nicotinamide pocket, including the regions bound to the nicotinamide ribose and phosphate, should inhibit NAD(H) binding and interfere with tetrameric assembly. We are using these results to guide our development of anti-neoplastic CtBP specific inhibitors.

C-terminal binding proteins (CtBP1 & 2) are co-transcriptional factors that have been implicated in progression of a number of cancers. CtBP possesses a D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domain that binds NAD(H) and provides an attractive target for small molecule intervention. Our crystal structures of CtBP1 and CtBP2 with bound substrate led to the design of an inhibitor (HIPP) with ∼300nm binding affinity. Experiments using HIPP in APCmin (colon cancer) mouse models suggest that CtBP inhibitors could be efficacious agents in a number of cancers. NAD(H) linked oligomeric assembly of CtBP has been suggested to contribute to co-transcriptional signaling, although the biologically relevant level of CtBP assembly has been unclear. Using multi-angle light scattering (MALS), we find that CtBP assembles from dimers into tetramers as a function of NAD(H) concentration with EC50 values in the range of 50-150 nM, depending on the construct used for CtBP1 and CtBP2. Although crystal structures of CtBP1 and CtBP2 have been described in terms of dimers, due to extensive intradimeric interactions, examination of the distinct crystal lattices for CtBP1 and CtBP2 reveal a very similar tetrameric assembly in both paralogs. Site-directed mutations of residues involved in the observed lattice contacts support the lattice tetramer as the solution tetramer. We propose that this tetramer is the co-transcriptionally active form of CtBP and are investigating inhibitors that disrupt this tetrameric assembly as potential lead compounds for a therapeutically useful CtBP specific inhibitor.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

1 Active-site tryptophan, the target of anti-neoplastic CtBP inhibitors, mediates inhibitor disruption of CtBP oligomerization and transcription coregulatory activities M. Michael Dcona, Priyadarshan K. Damle#, Francisco Zarate-Perez#, Benjamin L. Morris#, Zaid Nawaz, Michael J. Dennis, Xiaoyan Deng, Sudha Korwar, Sahib J. Singh, Keith C. Ellis, William E. Royer, Dipankar Bandyopadhyay, Carlos Escalante, and Steven R. Grossman * Departments of Internal Medicine (MMD, PKD, ZN, MJD, SRG), Human and Molecular Genetics (BLM, SRG), Physiology and Biophysics (FZP, CE), Medicinal Chemistry (SK, KCE), and Biostatistics (DB, XD), VCU Massey Cancer Center (KCE, DB, CE and SRG), Virginia Commonwealth University, Richmond, VA 23220, USA; Department of Biochemistry & Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605 (WER), USA. # Equal Contribution This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on April 29, 2019 as DOI: 10.1124/mol.118.114363

C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD+- and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate- and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.

Abstract C-terminal Binding Proteins (CtBP) 1 and 2 constitute a family of oncogenic transcriptional co-regulators overexpressed in tumor tissues that are associated with worse prognostic outcome and aggressive tumor characteristics in multiple cancer types. Specifically, CtBP has been found to repress expression of genes responsible for apoptosis and EMT (eg. BIK and CDH1) and promote expression of genes that partake in the migration of cancer cells and those that are responsible for enhanced drug resistance (eg. TIAM1 and MDR1). CtBP2 is also critically required for colon cancer stem cell self-renewal. CtBP is unique among transcription co-regulators in harboring a conserved D-isomer specific 2-hydroxyacid dehydrogenase (D2DH) domain, which reduces an alpha-keto acid substrate to an alpha-hydroxy acid in the presence of NADH. The presence of NADH also facilities oligomerization of CtBP, leading to assembly of higher order complexes of CtBP with both DNA binding transcription factors and histone modifying enzymes that then leads to modulation target genes. We have identified hydroxyimino-3-phenylpropanoic acid and its 4-chloro derivative (HIPP; 4-Cl-HIPP) as potent substrate competitive inhibitors of the CtBP dehydrogenase (IC50’s=240nM, 180 nM), which also disrupt CtBP oligomerization, promoter localization, and transcriptional regulation. Co-crystallization of HIPP and CtBP1/2 indicated a strong π-π interaction between the HIPP phenyl ring and the indolyl ring in tryptophan W318/324 of CtBP1/2, indicating that this tryptophan is critical to CtBP interaction with HIPP inhibitors, dehydrogenase function, and quaternary structure. Of note W318/324, though conserved in CtBP1/2, is unique among D2DH, suggesting that a better understanding of W318/324 role and function in CtBP structure and function is critical to optimizing design of inhibitors that targeting this evolutionary unique residue among dehydrogenases. To further elucidate the mechanism of action in catalysis and oligomerization, as well as functional importance for transcription of CtBP2 W324, we analyzed enzyme kinetics, oligomerization, transcriptional co-regulatory activity and cell migration in a series of CtBP2 W324 mutants overexpressed in breast and colon cancer cell lines with concomitant knockdown (siRNA) or knockout (CRISPR/Cas9) of CtBP2. Our data demonstrates that mutation of W324 abrogated dehydrogenase activity, oligomerization, transactivation of the validated CtBP target gene TIAM1 and induction of migration. In summary, our findings suggest that the W324 residue is critical for CtBP2’s function and its unique conservation in CtBP1/2 vs. other dehydrogenases will allow the development of high specificity CtBP W318/324 inhibitors to limit potential toxicity due to off target inhibition of related metabolic dehydrogenases. Citation Format: Martin M. Dcona, Benjamin L. Morris, Priyadarshan K. Damle, Zaid Nawaz, Francisco Zarate Perez, Michael J. Dennis, Sahib J. Singh, William E. Royer, Keith C. Ellis, Steven R. Grossman. Tryptophan 318/324, the target of C-terminal binding protein (CtBP) inhibitors, plays a critical role in CtBP enzymatic activity, oligomerization and transcriptional coregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3204. doi:10.1158/1538-7445.AM2017-3204

Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 Å. This structure not only visualizes the active site poised for catalysis of APOBEC3A, but pinpoints the residues that confer specificity towards CC/TC motifs. The APOBEC3A–ssDNA complex defines the 5′–3′ directionality and subtle conformational changes that clench the ssDNA within the binding groove, revealing the architecture and mechanism of ssDNA recognition that is likely conserved among all polynucleotide deaminases, thereby opening the door for the design of mechanistic-based therapeutics.

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD+ as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24μM). A structure–activity relationship study of this compound further identified the 4-chloro- (IC50=0.18μM) and 3-chloro- (IC50=0.17μM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

Abstract C-terminal binding proteins 1 and 2 (CtBP) are transcriptional coregulators whose overexpression has been linked to poor prognosis and/or chemoresistance in most common solid tumor types. CtBP exerts oncogenic activities through modulation of gene expression programs governing cell survival, epithelial/mesenchymal transitions, migration/invasion, and cell cycle, and is also required for colon cancer tumor initiating cell function. CtBP, however, has never been formally characterized as an oncogene. We now show that Ctbp2 exhibits transforming activity in immortalized NIH 3T3 as well as mouse and human primary cells. Ctbp2 alone, or in cooperation with SV40 large T antigen (LT) transformed NIH 3T3 cells and MEF’s, respectively, to anchorage independence with similar efficiency to mutant H-Ras. Human BJ foreskin fibroblasts were also transformed to anchorage independence by CtBP2 in cooperation with LT, SV40 small T antigen, and h-TERT with efficiency similar to H-Ras, indicating that CtBP overexpression, as found in the majority of human colon, ovary, breast, and prostate cancers, may be a key oncogenic driver gene. To confirm the physiologic role of Ctbp2 in a mouse tumor model with Ctbp overexpression, we bred Apcmin/+ mice to Ctbp2 hemizygote (Ctbp2+/-) mice, which are otherwise healthy. CtBP is a known target of the APC E3 ligase and is thus stabilized in APC mutated human colon cancers and is found in high levels in APCmin polyps. Remarkably, survival to humane endpoint at 37 weeks for Apcmin/+ vs. Apcmin/+-Ctbp2+/- mice was 0% vs. 100% (p <0.001). As CtBP also encodes a targetable intrinsic dehydrogenase that forms an NADH-dependent oligomerization surface, our cell and mouse model data supports the idea that CtBP could be an attractive small molecule drug target in tumors where it is upregulated. We have therefore synthesized a series of dehydrogenase substrate competitive inhibitor compounds based on CtBP's natural substrate 4-methylthio-2-oxobutyric acid (MTOB) that utilize a phenylpyruvate backbone and demonstrate nanomolar enzymatic IC50's with micromolar cellular GI50’s, while exhibiting minimal off target inhibition of lactate dehydrogenase. Phenylpyruvate—based CtBP inhibitors biochemically caused dissolution of CtBP oligomers, which are the active transcriptional conformation, and exhibited on-target transcriptional effects in cells, with reversal of CtBP transcriptional repression of key cancer target genes, such as E-cadherin, Bik, and BRCA1. In summary, we have characterized CtBP2 as a cellular proto-oncogene that can transform primary mouse and human cells in a manner similar to activated H-Ras, genetically validated Ctbp2 as a therapeutic target in the Apcmin/+ mouse model, and identify promising small molecule lead inhibitors for future therapeutic development. Citation Format: Evan T. Sumner, Sudha Korwar, Benjamin L. Morris, Martin M. Dcona, Brendan J. Hilbert, William E. Royer, Keith C. Ellis, Steven Grossman. C-terminal binding protein (CtBP): An emerging oncogene and small molecule drug target in solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2003.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB, and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

The oncogenic corepressors C‐terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d ‐isomer specific 2‐hydroxyacid dehydrogenase ( d 2‐HDH) domains. 4‐Methylthio 2‐oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD + revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD + phosphate. Neither feature is present in other d 2‐HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti‐neoplastic CtBP inhibitors.

The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII, Each AB dimeric half of HbII is assembled in a manner very similar to that of the well-studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels. Despite very similar ligand-linked structural transitions observed in HbI and HbII crystals, HbII in the crystal or encapsulated in silica gels apparently exhibits minimal cooperativity in oxygen binding, in contrast with the full cooperativity exhibited by HbI crystals. However, oxygen binding curves in the crystal indicate the presence of a significant functional inequivalence of A and B chains. When this inequivalence is taken into account, both crystal and R state gel functional data are consistent with the conservation of a tertiary contribution to cooperative oxygen binding, quantitatively similar to that measured for HbI, and are in keeping with the structural information. Furthermore, our results indicate that to fully express cooperative ligand binding, HbII requires quaternary transitions hampered by crystal lattice and gel encapsulation, revealing greater complexity in cooperative function than the direct communication across a dimeric interface observed in HbI.

Dynamic behavior of proteins is critical to their function. X-ray crystallography, a powerful yet mostly static technique, faces inherent challenges in acquiring dynamic information despite decades of effort. Dynamic `structural changes' are often indirectly inferred from `structural differences' by comparing related static structures. In contrast, the direct observation of dynamic structural changes requires the initiation of a biochemical reaction or process in a crystal. Both the direct and the indirect approaches share a common challenge in analysis: how to interpret the structural heterogeneity intrinsic to all dynamic processes. This paper presents a real-space approach to this challenge, in which a suite of analytical methods and tools to identify and refine the mixed structural species present in multiple crystallographic data sets have been developed. These methods have been applied to representative scenarios in dynamic crystallography, and reveal structural information that is otherwise difficult to interpret or inaccessible using conventional methods.