Unveiling the Unfolding Pathway of F5F8D Disorder-Associated D81H/V100D Mutant of MCFD2 via Multiple Molecular Dynamics Simulations

Combined factor deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in the LMAN1 or MCFD2 genes. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, EF-hand-Ca21 dissociation and assembly of misfolded MCFD2-LMAN1 complex. Here, we have investigated the solution conformations of the MCFD2((D81H,V100D)) protein mutant through extensive molecular dynamics (MD) simulations. The V100D, one of the many MCFD2 mutations known to be associated to F5F8D, is difficult to be reconciled with the pathway model because it is located far from the metal sites and the MCFD2/LMAN1 interface. Consequently, an inspection of all the steps involved in D81H/V100D MCFD2 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. A comparison with parallel studies carried out for the Wild-Type (WT) MCFD2 pointed out that the mutation decreases the affinity of the protein for the Ca21 ion. Multiple explicit solvents MD simulations (50_ns) performed on the two proteins revealed that in the WT protein, stable H-bond network and compact hydrophobic core region are created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. In fact it is shown that the V100D mutation, although located far away the EF-hand domain, may induce subtle modification in the structural core of MCFD2 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway. The native-like hydrophobic cluster formed near the V100 residue in the wild-type protein is disrupted by the negatively charged Asparagine residue. Furthermore, the presence of the D81H mutation in the EF-1 hand domain may also increase the protein unfolding rate and consequently prevent the formation of the MCFD2-LMAN1 complex. The detailed structural insights obtained from our large-scale simulations complement the clinical features and offer useful insights into the mechanism behind MCFD2 protein misfolding.     

Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)

LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca²⁺-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca²⁺ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca²⁺ concentrations. Our results suggest that compartmental changes in Ca²⁺ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.     

Crystal structure of the LMAN1-CRD/MCFD2 transport receptor complex provides insight into combined deficiency of factor V and factor VIII

LMAN1 is a glycoprotein receptor, mediating transfer from the ER to the ER-Golgi intermediate compartment. Together with the co-receptor MCFD2, it transports coagulation factors V and VIII. Mutations in LMAN1 and MCFD2 can cause combined deficiency of factors V and VIII (F5F8D). We present the crystal structure of the LMAN1/MCFD2 complex and relate it to patient mutations. Circular dichroism data show that the majority of the substitution mutations give rise to a disordered or severely destabilized MCFD2 protein. The few stable mutation variants are found in the binding surface of the complex leading to impaired LMAN1 binding and F5F8D.

Structured summary

MINT-7557086: lman1 (uniprotkb:P49257) and mcfd2 (uniprotkb:Q8NI22) bind (MI:0407) by X-ray crystallography (MI:0114)     

New insights into multiple coagulation factor deficiency from the solution structure of human MCFD2

Human MCFD2 (multiple coagulation factor deficiency 2) is a 16-kDa protein known to participate in transport of the glycosylated human coagulation factors V and VIII along the secretory pathway. Mutations in MCFD2 or in its binding partner, the membrane-bound transporter ERGIC (endoplasmic reticulum-Golgi intermediate compartment)-53, cause a mild form of inherited hemophilia known as combined deficiency of factors V and VIII (F5F8D). While ERGIC-53 is known to be a lectin-type mannose binding protein, the role of MCFD2 in the secretory pathway is comparatively unclear. MCFD2 has been shown to bind both ERGIC-53 and the blood coagulation factors, but little is known about the binding sites or the true function of the protein. In order to facilitate understanding of the function of MCFD2 and the mechanism by which mutations in the protein cause F5F8D, we have determined the structure of human MCFD2 in solution by NMR. Our results show the folding of MCFD2 to be dependent on availability of calcium ions. The protein, which is disordered in the apo state, folds upon binding of Ca²⁺ to the two EF-hand motifs of its C-terminus, while retaining some localized disorder in the N-terminus. NMR studies on two disease-causing mutant variants of MCFD2 show both to be predominantly disordered, even in the presence of calcium ions. These results provide an explanation for the previously observed calcium dependence of the MCFD2-ERGIC-53 interaction and, furthermore, clarify the means by which mutations in this protein result in inefficient secretion of blood coagulation factors V and VIII.     

LMAN1 and MCFD2 form a cargo receptor complex and interact with coagulation factor VIII in the early secretory pathway

Mutations in LMAN1 (ERGIC-53) and MCFD2 are the causes of a human genetic disorder, combined deficiency of coagulation factor V and factor VIII. LMAN1 is a type 1 transmembrane protein with homology to mannose-binding lectins. MCFD2 is a soluble EF-hand-containing protein that is retained in the endoplasmic reticulum through its interaction with LMAN1. We showed that endogenous LMAN1 and MCFD2 are present primarily in complex with each other with a 1:1 stoichiometry, although MCFD2 is not required for oligomerization of LMAN1. Using a cross-linking-immunoprecipitation assay, we detected a specific interaction of both LMAN1 and MCFD2 with factor VIII, with the B domain as the most likely site of interaction. We also present evidence that this interaction is independent of the glycosylation state of factor VIII but requires native calcium concentration in the endoplasmic reticulum. The interaction of MCFD2 with factor VIII appeared to be independent of LMAN1-MCFD2 complex formation. These results suggest that LMAN1 and MCFD2 form a cargo receptor complex and that the primary sorting signals residing in the B domain direct the binding of factor VIII to LMAN1-MCFD2 through calcium-dependent protein-protein interactions. MCFD2 may function to specifically recruit factor V and factor VIII to sites of transport vesicle budding within the endoplasmic reticulum lumen.     

A hidden aggregation‐prone structure in the heart of hypoxia inducible factor prolyl hydroxylase

Prolyl hydroxylase domain‐containing protein 2 (PHD2), as one of the most important regulators of angiogenesis and metastasis of cancer cells, is a promising target for cancer therapy drug design. Progressive studies imply that abnormality in PHD2 function may be due to misfolding. Therefore, study of the PHD2 unfolding pathway paves the way for a better understanding of the influence of PHD2 mutations and cancer cell metabolites on the protein folding pathway. We study the unfolding of the PHD2 catalytic domain using differential scanning calorimetry (DSC), fluorescence spectroscopy, and discrete molecular dynamics simulations (DMD). Using computational and experimental techniques, we find that PHD2 undergoes four transitions along the thermal unfolding pathway. To illustrate PHD2 unfolding events in atomic detail, we utilize DMD simulations. Analysis of computational results indicates an intermediate species in the PHD2 unfolding pathway that may enhance aggregation propensity, explaining mutation‐independent PHD2 malfunction. Proteins 2016; 84:611–623. © 2016 Wiley Periodicals, Inc.     

Thermal Stability and Unfolding Pathways of Sso7d and its Mutant F31A: Insight from Molecular Dynamics Simulation

Abstract

The thermo-stability and unfolding behaviors of a small hyperthermophilic protein Sso7d as well as its single-point mutation F31A are studied by molecular dynamics simulation at temperatures of 300 K, 371 K and 500 K. Simulations at 300 K show that the F31A mutant displays a much larger flexibility than the wild type, which implies that the mutation obviously decreases the protein's stability. In the simulations at 371 K, although larger fluctuations were observed, both of these two maintain their stable conformations. High temperature simulations at 500 K suggest that the unfolding of these two proteins evolves along different pathways. For the wild-type protein, the C-terminal alpha-helix is melted at the early unfolding stage, whereas it is destroyed much later in the unfolding process of the F31A mutant. The results also show that the mutant unfolds much faster than its parent protein. The deeply buried aromatic cluster in the F31A mutant dissociates quickly relative to the wild-type protein at high temperature. Besides, it is found that the triple-stranded antiparallel β-sheet in the wild-type protein plays an important role in maintaining the stability of the entire structure.     

Cross-linking of ΔF508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴     

Calumenin contributes to ER-Ca2+ homeostasis in bronchial epithelial cells expressing WT and F508del mutated CFTR and to F508del-CFTR retention

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca²⁺) homeostasis. In this study, we discovered that the Ca²⁺ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca²⁺ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca²⁺ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.     

Elevated expression of the V-ATPase D2 subunit triggers increased energy metabolite levels in KrasG12D-driven cancer cells

Mutations of the Ras oncogene are frequently detected in human cancers. Among Ras-mediated tumorigenesis, Kras-driven cancers are the most dominant mutation types. Here, we investigated molecular markers related to the Kras mutation, which is involved in energy metabolism in Kras mutant-driven cancer. We first generated a knock-in Kras^G12D cell line as a model. The genotype and phenotype of the Kras ^G12D-driven cells were first confirmed. Dramatically elevated metabolite characterization was observed in Kras ^G12D-driven cells compared with wild-type cells. Analysis of mitochondrial metabolite-related genes showed that two of the 84 genes in Kras ^G12D-driven cells differed from control cells by at least twofold. The messenger RNA and protein levels of ATP6V0D2 were significantly upregulated in Kras ^G12D-driven cells. Knockdown of ATP6V0D2 expression inhibited motility and invasion but did not affect the proliferation of Kras ^G12D-driven cells. We further investigated ATP6V0D2 expression in tumor tissue microarrays. ATP6V0D2 overexpression was observed in most carcinoma tissues, such as melanoma, pancreas, and kidney. Thus, we suggest that ATP6V0D2, as one of the V-ATPase (vacuolar-type H ⁺-ATPase) subunit isoforms, may be a potential therapeutic target for Kras mutation cancer.     

Different misfolding mechanisms converge on common conformational changes

Prion diseases are caused by misfolding and aggregation of the prion protein (PrP). Pathogenic mutations such as Y218N and E196K are known to cause Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease, respectively. Here we describe molecular dynamics simulations of these mutant proteins to better characterize the detailed conformational effects of these sequence substitutions. Our results indicate that the mutations disrupt the wild-type native PrP^C structure and cause misfolding. Y218N reduced hydrophobic packing around the X-loop (residues 165–171), and E196K abolished an important wild-type salt bridge. While differences in the mutation site led PrP mutants to misfold along different pathways, we observed multiple traits of misfolding that were common to both mutants. Common traits of misfolding included: 1) detachment of the short helix (HA) from the PrP core; 2) exposure of side chain F198; and 3) formation of a nonnative strand at the N-terminus. The effect of the E196K mutation directly abolished the wild-type salt bridge E196-R156, which further destabilized the F198 hydrophobic pocket and HA. The Y218N mutation propagated its effect by increasing the HB-HC interhelical angle, which in turn disrupted the packing around F198. Furthermore, a nonnative contact formed between E221 and S132 on the S1-HA loop, which offered a direct mechanism for disrupting the hydrophobic packing between the S1-HA loop and HC. While there were common misfolding features shared between Y218N and E196K, the differences in the orientation of HB and HC and the X-loop conformation might provide a structural basis for identifying different prion strains.     

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adR^FA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adR^PA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adR^PA receptor is masked by the F4adR^FA. The hypothesis that F4adR^FA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy–Weinberg equilibrium statistics. The F4adR^PA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abR^PA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abR^FA/F4acR⁺/F4adR^FA); A2 (F4abR^FA/F4acR⁺/F4adR^PA); B (F4abR^FA/F4acR⁺/F4adR⁻); C1 (F4abR^PA/F4acR⁻/F4adR^FA); C2 (F4abR^PA/F4acR⁻/F4adR^PA); D1 (F4abR⁻/F4acR⁻/F4adR^FA); D2 (F4abR⁻/F4acR⁻/F4adR^PA); E (F4abR⁻/F4acR⁻/F4adR⁻).     

Thermal stability and unfolding pathways of Sso7d and its mutant F31A: insight from molecular dynamics simulation

The thermo-stability and unfolding behaviors of a small hyperthermophilic protein Sso7d as well as its single-point mutation F31A are studied by molecular dynamics simulation at temperatures of 300 K, 371 K and 500 K. Simulations at 300 K show that the F31A mutant displays a much larger flexibility than the wild type, which implies that the mutation obviously decreases the protein's stability. In the simulations at 371 K, although larger fluctuations were observed, both of these two maintain their stable conformations. High temperature simulations at 500 K suggest that the unfolding of these two proteins evolves along different pathways. For the wild-type protein, the C-terminal alpha-helix is melted at the early unfolding stage, whereas it is destroyed much later in the unfolding process of the F31A mutant. The results also show that the mutant unfolds much faster than its parent protein. The deeply buried aromatic cluster in the F31A mutant dissociates quickly relative to the wild-type protein at high temperature. Besides, it is found that the triple-stranded antiparallel β-sheet in the wild-type protein plays an important role in maintaining the stability of the entire structure.     

Pathogenic mutations in the hydrophobic core of the human prion protein can promote structural instability and misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases.     

A missense mutation in LRR8 of RXFP2 is associated with cryptorchidism

Using genome-wide mutagenesis with N-ethyl-N-nitrosourea (ENU), a mouse mutant with cryptorchidism was identified. Genome mapping and exon sequencing identified a novel missense mutation (D294G) in Relaxin/insulin-like family peptide receptor 2 (Rxfp2). The mutation impaired testicular descent and resulted in decreased testis weight in Rxfp2 ^DG/DG mice compared to Rxfp2 ^+/DG and Rxfp2 ^+/+ mice. Testicular histology of the Rxfp2 ^DG/DG mice revealed spermatogenic defects ranging from germ cell loss to tubules with Sertoli-cell-only features. Genetic complementation analysis using a loss-of-function allele (Rxfp2 ^?) confirmed causality of the D294G mutation. Specifically, mice with one of each mutant allele (Rxfp2 ^DG/?) exhibited decreased testis weight and failure of the testes to descend compared to their Rxfp2 ^+/? littermates. Total and cell-surface expression of mouse RXFP2 protein and intracellular cAMP accumulation were measured. Total expression of the D294G protein was minimally reduced compared to wild-type, but cell-surface expression was markedly decreased. When analyzed for cAMP accumulation, the EC50 was similar for cells transfected with wild-type and mutant RXFP2 receptor. However, the maximum cAMP response that the mutant receptor reached was greatly reduced compared to the wild-type receptor. In silico modeling of leucine rich repeats (LRRs) 7–9 indicated that aspartic acid 294 is located within the β-pleated sheet of LRR8. We thus postulate that mutation of D294 results in protein misfolding and aberrant trafficking. The ENU-induced D294G mutation underscores the role of the INSL3/RXFP2-mediated pathway in testicular descent and expands the repertoire of mutations known to affect receptor trafficking and function.     

Cargo selectivity of the ERGIC-53/MCFD2 transport receptor complex

Exit of soluble secretory proteins from the endoplasmic reticulum (ER) can occur by receptor-mediated export as exemplified by blood coagulation factors V and VIII. Their efficient secretion requires the membrane lectin ER Golgi intermediate compartment protein-53 (ERGIC-53) and its soluble luminal interaction partner multiple coagulation factor deficiency protein 2 (MCFD2), which form a cargo receptor complex in the early secretory pathway. ERGIC-53 also interacts with the two lysosomal glycoproteins cathepsin Z and cathepsin C. Here, we tested the subunit interdependence and cargo selectivity of ERGIC-53 and MCFD2 by short interference RNA-based knockdown. In the absence of ERGIC-53, MCFD2 was secreted, whereas knocking down MCFD2 had no effect on the localization of ERGIC-53. Cargo binding properties of the ERGIC-53/MCFD2 complex were analyzed in vivo using yellow fluorescent protein fragment complementation. We found that MCFD2 is dispensable for the binding of cathepsin Z and cathepsin C to ERGIC-53. The results indicate that ERGIC-53 can bind cargo glycoproteins in an MCFD2-independent fashion and suggest that MCFD2 is a recruitment factor for blood coagulation factors V and VIII.     

Hydrophobic Core Mutations Associated with Cataract Development in Mice Destabilize Human ��D-Crystallin

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.     

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

The apolipoprotein E family contains three major isoforms (ApoE4, E3, and E2) that are directly involved with lipoprotein metabolism and cholesterol transport. ApoE3 and apoE4 differ in only a single amino acid with an arginine in apoE4 changed to a cysteine at position 112 in apoE3. Yet only apoE4 is recognized as a risk factor for Alzheimer''s disease. Here we used ¹⁹F NMR to examine structural differences between apoE4 and apoE3 and the effect of the C-terminal domain on the N-terminal domain. After incorporation of 5-¹⁹F-tryptophan the 1D ¹⁹F NMR spectra were compared for the N-terminal domain and for the full length proteins. The NMR spectra of the N-terminal region (residues 1–191) are reasonably well resolved while those of the full length wild-type proteins are broad and ill-defined suggesting considerable conformational heterogeneity. At least four of the seven tryptophan residues in the wild type protein appear to be solvent exposed. NMR spectra of the wild-type proteins were compared to apoE containing four mutations in the C-terminal region that gives rise to a monomeric form either of apoE3 under native conditions (Zhang et al., Biochemistry 2007; 46: 10722–10732) or apoE4 in the presence of 1 M urea. For either wild-type or mutant proteins the differences in tryptophan resonances in the N-terminal region of the protein suggest structural differences between apoE3 and apoE4. We conclude that these differences occur both as a consequence of the Arg158Cys mutation and as a consequence of the interaction with the C-terminal domain.     

The interplay between protein stability and dynamics in conformational diseases: The case of hPGK1 deficiency

Conformational diseases often show defective protein folding efficiency in vivo upon mutation, affecting protein properties such as thermodynamic stability and folding/unfolding/misfolding kinetics as well as the interactions of the protein with the protein homeostasis network. Human phosphoglycerate kinase 1 (hPGK1) deficiency is a rare inherited disease caused by mutations in hPGK1 that lead to loss-of-function. This disease offers an excellent opportunity to explore the complex relationships between protein stability and dynamics because of the different unfolding mechanisms displayed towards chemical and thermal denaturation. This work explores these relationships using two thermostable mutants (p.E252A and p.T378P) causing hPGK1 deficiency and WT hPGK1 using proteolysis and chemical denaturation. p.T378P is degraded ~ 30-fold faster at low protease concentrations (here, the proteolysis step is rate-limiting) and ~ 3-fold faster at high protease concentrations (where unfolding kinetics is rate-limiting) than WT and p.E252A, indicating that p.T378P is thermodynamically and kinetically destabilized. Urea denaturation studies support the decrease in thermodynamic stability and folding cooperativity for p.T378P, as well as changes in folding/unfolding kinetics. The present study reveals changes in the folding landscape of hPGK1 upon mutation that may affect protein folding efficiency and stability in vivo, also suggesting that native state stabilizers and protein homeostasis modulators may help to correct folding defects in hPGK1 deficiency. Moreover, detailed kinetic proteolysis studies are shown to be powerful and simple tools to provide deep insight into mutational effects on protein folding and stability in conformational diseases.